Close linkage of the olfactory marker protein gene to the mouse deafness mutation shaker-1. One thousand sixty-six progeny have been generated from a backcross segregating for the mouse deafness mutation, shaker-1 (sh-1). One thousand fifty-two mice were analyzed for a protein polymorphism segregating for the distal flanking marker, beta-globin (Hbb), and 13 recombinants between Hbb and sh-1 were identified. One thousand eight mice were analyzed for a restriction fragment length polymorphism segregating for the proximal flanking marker, tyrosinase (c), and 54 recombinants between c and sh-1 were identified, completing a panel of 67 recombinant mice from the backcross in the vicinity of the sh-1 mutation. This panel allows the identification of markers closely linked to the sh-1 mutation that may act as start points for a chromosomal walk to the gene. One such marker, the olfactory marker protein gene (Omp), is recombinant with sh-1 in only one mouse from the recombinant panel. Thus, the Omp gene lies 0.1 cM from sh-1, on average, a distance of 200 kb. Haplotype analysis indicates that Omp lies proximal to sh-1.


The D4 dopamine receptor (DRD4) maps to distal 11p close to HRAS. Dopaminergic pathophysiology is important in several psychiatric illnesses. The recently cloned D4 dopamine receptor gene (DRD4) shows considerable homology to the D2 and D3 dopamine receptors (DRD2 and DRD3); pharmacologically, its affinity for the atypical antipsychotic clozapine is much higher than that of these other dopamine receptors. Probe pB28 for this locus recognizes an informative HincII polymorphism. We typed this polymorphism on several large reference families (a total of about 271 individuals) to place DRD4 in the genetic linkage map. Pairwise linkage analysis (using ILINK) provided evidence for close linkage to the distal 11p loci tyrosine hydroxylase (TH) and the Harvey ras oncogene (HRAS). We used our version of LINKMAP adapted to run under distributed parallel processing (Linda-LINKMAP) for an analysis moving DRD4 across a fixed map with HRAS set 3.8 cM distal to TH. This localized DRD4 close to HRAS, with no crossovers observed between those loci and a maximum lod score of 19.9 (2 cM distal to HRAS). The one LOD unit support interval extends from about 1 cM proximal to HRAS to 8 cM distal to HRAS. Crossovers identified in one kindred place DRD4 distal to TH, providing further evidence for its location close to HRAS, making DRD4 one of the most telomeric of 11p markers. (This also places DRD4 in band 11p15.5.)


A glycine-to-glutamate substitution abolishes alanine:glyoxylate aminotransferase catalytic activity in a subset of patients with primary hyperoxaluria type 1. We have synthesized and sequenced alanine:glyoxylate aminotransferase (AGT; HGMW-approved symbol for the gene--AGXT) cDNA from the liver of a primary hyperoxaluria type 1 (PH1) patient who had normal levels of hepatic peroxisomal immunoreactive AGT protein, but no AGT catalytic activity. This revealed the presence of a single point mutation (G----A at cDNA nucleotide 367), which is predicted to cause a glycine-to-glutamate substitution at residue 82 of the AGT protein. This mutation is located in exon 2 of the AGT gene and leads to the loss of an AvaI restriction site. Exon 2-specific PCR followed by AvaI digestion showed that this patient was homozygous for this mutation. In addition, three other PH1 patients, one related to and two unrelated to, but with enzymological phenotype similar to that of the first patient, were also shown to be homozygous for the mutation. However, one other phenotypically similar PH1 patient was shown to lack this mutation. The mechanism by which the glycine-to-glutamate substitution at residue 82 causes loss of catalytic activity remains to be resolved. However, the protein sequence in this region is highly conserved between different mammals, and the substitution at residue 82 is predicted to cause significant local structural alterations.


Identification of three novel PKU mutations among Chinese: evidence for recombination or recurrent mutation at the PAH locus. Three novel mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese classical phenylketonuria (PKU) patients. Two of these substitutions (W326X and Y356X) result in the generation of a premature stop codon, while the third (IVS-7nt2) alters an invariant dinucleotide splicing signal. These mutations together account for about 10% of all PKU alleles in the Chinese population. The W326X mutation is associated with PAH RFLP haplotype 4, the most common haplotype in Orientals, while the IVS-7nt2 mutation occurs once on a haplotype 7 chromosome. The Y356X mutation is associated with multiple haplotypes, possibly due to crossover, gene conversion, or recurrent mutation.


Characterization of radiation/fusion hybrids containing parts of human chromosome 10 and their use in mapping chromosome 10-specific probes. We have characterized a panel of somatic cell hybrid cell lines which contain different portions of human chromosome 10. Genomic DNA from the somatic cell hybrids was tested for hybridization with each of an ordered set of probes used previously to construct a genetic map of chromosome 10, as well as several additional probes, previously localized by in situ hybridization. Hybridization of an unmapped probe to the cell line DNAs can be used to determine its most likely position on the chromosome relative to the mapped set of probes. Genomic DNA from two of the cell lines has been used to construct region-specific cosmid and bacteriophage libraries, and clones derived from these libraries were localized by hybridization to the panel of hybrid cell lines. Several of these probes reveal restriction fragment length polymorphisms which have been genetically mapped. Three of the probes map near the locus for multiple endocrine neoplasia type 2A, and one of these probes, BG-JC353 (D10S167), maps between RBP3 and TB14.34 (D10S34). Another probe, CRI-J282 (D10S104), is close to the FNRB locus. The panel of hybrid cell lines is thus useful for rapidly localizing unmapped probes and as a source of DNA for the construction of recombinant libraries derived from specific regions of the chromosome.


Human-mouse homologies in the region of the polycystic kidney disease gene (PKD1). Autosomal dominant polycystic kidney disease (PKD1) is linked to the alpha-globin locus near the telomere of chromosome 16p. We established the existence of a conserved linkage group in mouse by mapping conserved sequences and cDNAs from the region surrounding the PKD1 gene in the mouse genome. Results obtained with the BXD recombinant strain system and somatic cell hybrids show the homologous region to be located on mouse chromosome 17 near the globin pseudogene Hba-ps4, an unprocessed alpha-like globin gene. The markers we mapped are widely distributed over the region known to contain the PKD1 gene, and it is therefore likely that the mouse homologue of PKD1 is also located on mouse chromosome 17.


Wobbler, a mutation affecting motoneuron survival and gonadal functions in the mouse, maps to proximal chromosome 11. The wobbler mouse (genotype wr/wr) has been considered as an animal model for human neurodegenerative disorders. In the homozygous condition, the autosomal mutation wobbler (wr) causes a motoneuron disease and gonadal dysfunction. We have genetically mapped the wr gene, using an interspecific backcross between the laboratory strain C57BL/6J (wr/+) and Mus spretus. The expected percentage of wobbler progeny were obtained, but heterogeneous expression of the wobbler phenotype indicated the existence of modifier genes in the M. spretus genetic background. The segregation of DNA markers of known chromosomal location among wobbler progeny and unaffected mice was scored. Close linkage of wr was obtained with Erbb and Rel on chromosome 11 and the gene order cen-Nfh-Erbb-wr-Rel-Hba-Il-3 was established. Closely linked markers like Erbb provide tools for a prognostic DNA diagnosis of the wobbler disease, and thereby for its analysis by descriptive and experimental embryology.


A minisatellite and a microsatellite polymorphism within 1.5 kb at the human muscle glycogen phosphorylase (PYGM) locus can be amplified by PCR and have combined informativeness of PIC 0.95. We sequenced a genomic clone (pMCMP1), previously reported to detect a VNTR polymorphism at the PYGM locus, and found a dinucleotide repeat segment (CA)14(GA)25 and a complex (AT)-repeat-rich segment containing 63 repeats spanning 160 bp. Resolution of PCR-amplified genomic DNA from the (CA)(GA) repeat region on DNA sequencing gels revealed a highly informative polymorphism with alleles differing by 2-bp intervals and ranging in size from 156 to 190 bp. Among three racial groups, a total of 18 alleles were observed. Fourteen alleles were observed in Caucasians (PIC 0.89), 12 alleles in American Blacks (PIC 0.89), and 9 alleles in Pima Indians (PIC 0.73). PCR amplification of the (AT) repeat region and resolution of the products on DNA sequencing gels revealed a complex variable length polymorphism with alleles distributed in size from 367 to 970 bp. Twenty-eight alleles were found in American Blacks (PIC 0.94), 6 alleles in Pima Indians (PIC 0.70), and 11 alleles in Caucasians (PIC 0.71). Comparison of the previously described VNTR RFLP alleles visualized by Southern hybridization to the PCR products described in this report demonstrated that the polymorphism described in both assays was identical. However, a larger number of alleles could be detected from the PCR-amplified products. Combined informativeness, PIC 0.95, for the two polymorphisms was determined from haplotype analysis of 100 Caucasian chromosomes. Therefore, for genotyping purposes, informativeness is maximized from using both polymorphisms.


Amplification of human polymorphic sites in the X-chromosomal region q21.33 to q24: DXS17, DXS87, DXS287, and alpha-galactosidase A. Methods for the PCR amplification of five polymorphic sites in the region Xq21.33 to Xq24 were developed and used to predict heterozygosity for Fabry disease in informative families. Clones containing polymorphic sites associated with DNA segments DXS17, DXS87, and DXS287, and the alpha-galactosidase A gene were isolated from genomic libraries. Surrounding nucleotide sequences and optimal conditions for amplification of each polymorphic site were determined. These amplifiable polymorphisms provided predictions of heterozygosity for Fabry disease and should be useful for diagnostic linkage analyses in Alport syndrome, X-linked cleft palate and ankyloglossia, Pelizaeus-Merzbacher disease, and X-linked agammaglobulinemia as well as sequence-tagged sites for gene mapping.


An approach to the localization of the susceptibility genes for generalized myasthenia gravis by mapping recombinant ancestral haplotypes. The association of HLA A1, B8, and DR3 with generalized myasthenia gravis (GMG) in Caucasoids is well established, but no particular gene has been implicated and there is still no adequate explanation in functional terms. In this study we have taken advantage of sequential genomic markers between B8 and DR3 so as to map the location of susceptibility gene(s) on the A1, B8, DR3 (8.1) ancestral haplotype. By studying 51 patients, we have delineated a region between HLA B and TNF which is shared by 29/29 patients with B8 and DR3, 19/19 patients with B8 but not DR3 and 2/3 patients with DR3 but not B8. The potential importance of this region was confirmed by examining a similar disease induced by D-Penicillamine (D-PenMG) and associated with different HLA class II alleles (DR1 and/or DR7). Among these patients, 7/16 (44%) have B8, often with other markers of 8.1. These results implicate at least two categories of genes in determining susceptibility to MG; one located in the region between HLA B and TNF may be immunoregulatory, whereas the second, located in the class II region, may relate to the inducing factor (e.g., DR1 or DR7 in D-PenMG).


Genomic DNA sequence and organization of a TL-like gene in the grc-G/C region of the rat. Genes in the grc-G/C region, which is linked to the rat major histocompatibility complex, influence the control of growth, development, and susceptibility to chemical carcinogens. As an initial approach to analyzing the structure and organization of these genes, a class I hybridizing fragment designated RT(5.8) was isolated from an R21 genomic DNA library and sequenced from overlapping restriction enzyme fragments. The RT(5.8) clone has 5788 base pairs and contains the eight exons characteristic of a class I gene. There are CAAT and TATA boxes upstream of the signal peptide, and the recognition sequence that precedes the site of polyadenylation is located downstream from the third cytoplasmic domain. Comparison of the RT(5.8) gene with representative class I genes from the rat and other species shows that the nucleotide sequences of RT(5.8) have a high level of similarity to those of TL region genes of several strains of mice. The peptide sequence deduced from the RT(5.8) clone is distinct from all previously published class I gene sequences, and at many positions there are amino acid residues that are unique to the RT(5.8) sequence. Probes have been isolated from the third exon and from the 5' and 3' flanking regions of the RT(5.8) clone, and Southern blot analysis with genomic DNA of various rat strains shows that these probes are specific for the RT(5.8) fragment. Northern blot analysis shows that the gene is transcribed in the thymus but not in the liver or spleen. The RT(5.8) sequence is more similar to some mouse TL genes (especially in the alpha 2 and cytoplasmic domains and in the 5' and 3' untranslated regions) than it is to other rat class I genes. Hence, TL-like genes are not restricted to the mouse.


Synthesis and release of neuroactive substances by glial cells. Glia contain, synthesize, or release more than 20 neuroactive compounds including neuropeptides, amino acid transmitters, eicosanoids, steroids, and growth factors. The stimuli that elicit release differ among compounds but include neuropeptides, neurotransmitters, receptor agonists, and elevated external [K+]. The mechanisms of release are poorly understood in most cases. Many of the neuroactive compounds are localized in discrete subpopulations of glia. Thus, glia are equipped to send as well as receive chemical messages and appear to be present as classes of cells with differing abilities to communicate chemically. It is possible that glia are as diverse as neurons in their functional characteristics.


Spontaneous changes in intracellular calcium concentration in type I astrocytes from rat cerebral cortex in primary culture. Measurement of fura-2 fluorescence in type I astrocytes from rat cerebral cortex showed that the intracellular calcium ion concentration undergoes very large spontaneous changes. These spike-like changes ranged from resting levels of calcium of 50-250 nM to as high as 1-2 microM. The spikes were found to be irregular in frequency and amplitude and were frequently synchronous in confluent cultures. The synchronous events appeared as propagating waves that spread over many cells. The spontaneous spikes persisted when the extracellular calcium concentration was reduced to below micromolar levels suggesting that the source for the increases in [Ca2+]i was intracellular. Treatment of the astrocytes with tetrodotoxin did not abolish the spontaneous changes, nor did blockade of voltage-dependent calcium channels with nimodipine and D-600. Ryanodine, a blocker of the sarcoplasmic reticulum calcium-induced calcium release channel, was also without effect. These changes in [Ca2+]i were different in character from both agonist-induced oscillations and depolarization-induced increases in intracellular calcium concentration. Depolarization using 25-100 mM [K+]o resulted in a prompt rise in intracellular calcium concentration, which returned to near resting levels, and this response was sensitive to removal of extracellular calcium and voltage-gated calcium channel antagonists. L-glutamate (0.5-100 microM) caused large increases in [Ca2+]i that were associated with discrete periodic oscillations in some cells. The cellular trigger for the spontaneous spikes is currently not understood. We conclude that spontaneous changes in [Ca2+]i in astrocytes are distinct from agonist-induced and membrane potential depolarization-induced changes.


Influence of serum progesterone levels at the time of hCG on the release of ova during hMG cycles. A study was designed to monitor release of ova by sonography in hMG-treated patients following hCG and to determine if failure to release ova correlates with critically low or high progesterone levels. This was a retrospective study of 292 consecutive patients treated with hMG. The requirement for treatment was that hCG be given when at least one follicle attained a 17-mm diameter with a serum estradiol level of at least 200 pg/mL per mature follicle. If the serum progesterone assay was greater than or equal to 1.8 ng/mL, then hCG would be given as long as there was at least one dominant follicle and a serum estradiol level greater than 200 pg/mL. The patients were divided into four groups for study based on the progesterone level at the time of hCG administration. There were no statistically significant differences in the ability to achieve ova release whether serum progesterone was very low or close to 2 ng/mL when hCG was given. The rise in the progesterone level prior to ovulation has been proposed to enhance egg release. However, the data presented herein do not support the necessity for a critical level of serum progesterone at the time of hCG injection in hMG-treated women.


Transcervical fallopian tube recanalization: a safe and effective therapy for patients with proximal tubal obstruction. Over a 13-month period, 14 patients with proximal tubal obstruction underwent transcervical fallopian tube recanalization under fluoroscopic guidance in an outpatient setting at the hospital of the University of Pennsylvania. Twenty-one of 24 attempted tubal dilations (87.5%) were successful, as demonstrated by tubal opacification and contrast spillage into the peritoneal cavity at the conclusion of the procedure. Four intrauterine pregnancies, and no ectopic pregnancies, have followed the recanalization. One pregnancy ended in an early miscarriage, one patient delivered a healthy term female, and two pregnancies are ongoing at greater than twenty weeks' gestation. Two procedure-related complications occurred: in one patient, the isthmic segment of a fallopian tube was perforated, but healed without incident, and another patient experienced a low-grade fever, which resolved with p.o. antibiotics. We therefore conclude that fallopian tube recanalization is a well-tolerated, safe, and effective procedure for the treatment of proximal tubal occlusion.


Further evidence concerning catecholamine metabolism in polycystic ovary syndrome following Gn-RH administration. The role of catecholamine activity in the GnRH-LH system among cases of polycystic ovary syndrome was investigated by high-performance liquid chromatography using an electrochemical detector. We measured plasma levels of catecholamine metabolites, 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenylacetic acid (DOPAC), in relation to LH-RH administration in 23 women with polycystic ovary syndrome. MHPG concentrations were significantly higher in polycystic ovary syndrome patients than in ten control subjects in early follicular phase. The peak values of LH after LH-RH administration were significantly correlated with plasma MHPG concentrations. These data suggest that an excess of norepinephrine is present in polycystic ovary syndrome and that the hypophyseal response to LH-RH administration is correlated with catecholamine, especially norepinephrine activity.


Comparison of the adrenocorticotropic hormone stimulation test in the follicular and luteal phases of the menstrual cycle. A comparison was made of the ACTH stimulation test in the proliferative and luteal phases of the menstrual cycle in 13 subjects, each of whom served as her own control. The test yielded a 53.9% false-positive rate in the luteal phase. The study, therefore, clearly shows that from a practical, clinical point of view, the test is completely unreliable in the luteal phase, and is only valid when carried out in the follicular phase of the cycle.


Annual and sub-annual rhythms in human conception rates. I. Effective correction and use of public record LMP dates. Most studies of rhythms in human conception rates have used monthly total births back-dated 9 months. Such data are of little use for studying any pattern less than a few months in length. Even at that level, they are poor material for studying the possibility of altered conception rhythms in anomalous births. The high frequency of premature delivery in such births makes a simple 9-month offset simplistic and misleading. Dates of last normal menses (LMP) provide appropriate detail, but have generally been dismissed as unreliable. In a large public birth record dataset, we have identified a clear artifact of poor LMP recall, highly correlated with passage of time between conception and onset of prenatal care. The majority of the variation in daily LMP counts due to this recall artifact can be corrected statistically to yield population data suitable to more detailed analyses.


Argon laser laparoscopy: an effective technique for conservative treatment of unruptured ectopic pregnancy. Between October 1987 and January 1990, 60 patients with unruptured ectopic pregnancy in the ampullary portion of the tube were treated with linear salpingotomy, using the argon laser laparoscopically. In all cases, salpingotomy was performed by the "bare-fiber" technique, with a power of 3-6 watts. The same fiber was used for incision and coagulation either with a contact or a non-contact technique. There were no instrument-related intra- or postoperative complications. Twenty-four patients were evaluated by second-look laparoscopy or hysterosalpingography for tube patency and possible formation of adhesions. In all of these patients the tubes were patent. In one patient, the tube failed to close, resulting in a fistula. Four patients had filmy adhesions, covering the tube near the incision site. There were no adhesions covering the distal part of the tube.


Follicular phase hormonal profiles during administration of leuprolide for in vitro fertilization. Hormonal changes induced during short-term administration of leuprolide were evaluated during the follicular phase in 57 patients who completed an IVF cycle. They were compared with those of 14 patients who were placed on long-term suppression. There was an unexpected abnormal increase in serum progesterone during the first week of the cycle in nine of the 57, with no significant change in the fertilization and cleavage rate; however, no pregnancy was achieved in this group. Transient mild elevation of progesterone was also detected in 16 patients with no adverse effect on fertilization and the outcome of the IVF. In the long protocol, the tonic levels of LH, FSH, and progesterone remained low throughout the follicular phase. The total number of pregnancies was higher in the short suppression regimen, but the full-term pregnancy rates were similar in both protocols.


Measurement of cell proliferation in gastric carcinoma: comparative analysis of Ki-67 and proliferative cell nuclear antigen (PCNA). Immunostaining to identify nuclear antigens expressed throughout the cell cycle provides a convenient way of assessing proliferating kinetics in tumours. We studied proliferation activity of gastric carcinomas by Ki-67 and PCNA immunostaining and the two methods were compared. The mode of tissue preparation differed, fresh frozen for Ki-67 and formalin-fixed paraffin-embedded for PCNA. Immunostaining with avidin-biotin was used in both. The labelling index (LI) and a semi-quantitative grading of cell proliferation were assessed in both markers. Significant correlation was shown between LI and grading with either Ki-67 and PCNA. However, no correlation was found between PCNA and Ki-67. This lack of relationship between the two markers may be attributed to a number of factors. 1. The most likely is the marked inter- and intra-tumour heterogeneity of gastric carcinomas reflected in high standard deviation values. 2. Preparation of tissue and small size sampling with Ki-67. 3. Long life of PCNA leading to detection of cells that have recently left the cell cycle. 4. One may be observing deregulated expression of DNA as seen in certain tumours. PCNA offers the advantage of being applicable to archival material.


Coexpression of UmuD' with UmuC suppresses the UV mutagenesis deficiency of groE mutants. The GroE proteins of Escherichia coli are heat shock proteins which have also been shown to be molecular chaperone proteins. Our previous work has shown that the GroE proteins of E. coli are required for UV mutagenesis. This process requires the umuDC genes which are regulated by the SOS regulon. As part of the UV mutagenesis pathway, the product of the umuD gene, UmuD, is posttranslationally cleaved to yield the active form, UmuD'. In order to investigate what role the groE gene products play in UV mutagenesis, we measured UV mutagenesis in groE+ and groE strains which were expressing either the umuDC or umuD'C genes. We found that expression of umuD' instead of umuD will suppress the nonmutability conferred by the groE mutations. However, cleavage of UmuD to UmuD' is unaffected by mutations at the groE locus. Instead we found that the presence of UmuD' increased the stability of UmuC in groE strains. In addition, we obtained evidence which indicates that GroEL interacts directly with UmuC.


Ultrastructural features of round-headed human spermatozoa. Two cases of the round-headed spermatozoa syndrome are described, with the main emphasis on the ultrastructural features of the spermatozoa. Essentially, the first type (type I) of these cells has round heads, lacks an acrosome and its intrinsic enzymes, and lacks a postacrosomal sheath; patients with this condition are absolutely infertile. This is the classical Schirren-Holstein model for the round-headed spermatozoon. Other types of round-headed spermatozoa might exist (Type II), which may possess remnants of the acrosome and are capable of fertilisation.


The effect of insulin on in vitro progesterone production of human granulosa cells. Insulin appears to play an important role in regulating ovarian function in some mammals. The present study assessed the effect of insulin on progesterone production by human granulosa lutein (G-L) cells in vitro. G-L cells were isolated from individual follicles at the time of laparoscopy for oocyte retrieval in patients undergoing in vitro fertilization/embryo transfer (IVF/ET). G-L cells were purified and plated for culture. Control wells contained no additions, while insulin was added to test wells. Although G-L cells obtained from follicles which contain an oocyte that subsequently fertilizes in vitro produce more progesterone at three and six days of culture than G-L cells from follicles that contain an oocyte that does not fertilize in vitro, insulin is unable to increase further the progesterone production of G-L cells from follicles containing either fertilized or nonfertilized oocytes at days 3 and 6 in culture. Further analysis based on the stimulation protocol (follicle stimulating hormone, n = 13; clomiphene citrate/hMG, n = 19; hMG, n = 10) also failed to yield a significant difference in basal or insulin-stimulated progesterone secretion after three or six days in culture. The lack of an effect of insulin on progesterone production by G-L cells in vitro may indicate that these cells are maximally stimulated following hyperstimulation and cannot increase progesterone production further, or may signify that insulin has no effect on the in vitro luteinization of human G-L cells obtained from hyperstimulated cycles.


Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum. The groESL operon of Clostridium acetobutylicum was cloned in Escherichia coli by using a gene probe of E. coli groESL. Sequencing of a positively reacting 2.2-kbp HindIII fragment contained in the recombinant plasmid pFN1 and a 2.5-kbp XbaI fragment present in pFN4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome. Two complete open reading frames (288 and 1632 bp) were found and identified as the groES- and groEL-homologous genes of C. acetobutylicum, respectively. The 3' end of a third gene (orfZ), which was divergently transcribed, showed no significant homology to other sequences available in the EMBL and GenBank data bases. The length of the groESL-specific mRNA (2.2 kb), a transcription terminator downstream of groEL, and a transcription start site upstream of groES, identified by primer extension analysis, indicated that groES and groEL of C. acetobutylicum are organized in a bicistronic operon. From the transcription start site, the promoter structure 5'-TTGCTA (17 bp) TATTAT that shows high homology to the consensus promoter sequence of gram-positive bacteria as well as E. coli was deduced. Transcription of the groESL operon was strongly heat inducible, and maximum levels of mRNA were detected 15 min after heat shock from 30 to 42 degrees C. An 11-bp inverted repeat, located between promoter and translation start sites of groES and partially identical with similar structures in front of several heat shock genes of other bacteria, may play an important role in the regulation of heat shock gene expression in this organism.


Electrostatic interactions stabilizing ferredoxin electron transfer complexes. Disruption by "conservative" mutations. Mitochondrial ferredoxins mediate electron transfer from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes. Previous studies on human ferredoxin, in which acidic residues were replaced with neutral amino acids, established that Asp-76 and Asp-79 are are important for binding to both reductase and P450 (Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612). Here we report that replacement of Asp----Glu at position 76 or 79, whereas maintaining negative charge at these positions also results in dramatic decreases in binding affinity for both electron transfer partners (5-100-fold, delta(delta G) approximately 1.0-2.8 kcal/mol). These results imply that the active electron transfer complexes in these systems are dominated by a stable form which requires specific pairwise electrostatic interactions of fixed geometry for recognition and binding. This mechanism contrasts with that proposed for other electron transfer systems (as exemplified by cytochrome c) in which electrostatic interactions are believed to function primarily in precollisional orientation leading to "encounter complexes" having multiple geometries of similar free energy.


Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo. We have examined the role of the acidic residues Asp2 and Glu4 at the NH2 terminus of Saccharomyces cerevisiae actin through site-directed mutagenesis. In DNEQ actin, these residues have been changed to Asn2 and Gln4, whereas in delta DSE actin, the Asp2-Ser-Glu tripeptide has been deleted. Both mutant actins can replace wild type yeast actin. Peptide mapping studies reveal that DNEQ, like wild type actin, retains the initiator Met and is NH2 terminally acetylated, whereas delta DSE has a free NH2 terminus and has lost the initiator Met. Interestingly, microscopic examination of filaments of these two actins reveal the appearance of bundled filaments. The DNEQ bundles are smaller and more ordered, whereas the delta DSE bundles are larger and more loosely organized. Additionally, both mutant actins activate the ATPase activity of rabbit muscle myosin S1 fragment to a lesser extent than wild type. We have also developed a sensitive assay for actin function in vivo that enabled us to detect a slight defect in the ability of these mutant actins to support secretion, an important function in yeast. Thus, although the mutant actins resulted in no gross phenotypic changes, we were able to detect a defect in actin function through this assay. From these studies we can conclude that 1) although NH2-terminal negative charges are not essential to yeast life, the loss of such charges does result in a slight defect in the actins' ability to support secretion, 2) removal of the NH2-terminal negative charges promotes the bundling of actin filaments, and 3) actins lacking NH2-terminal negative charges are unable to activate the myosin S1 ATPase activity as well as wild type actin.


Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides. Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) mini-exon is placed at the 5' end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5'-5') triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m7G(5')ppp(5')m6(2)AmpAmpCmpm3Ump. The presence of N6,N6,2'-O-trimethyladenosine and 3,2'-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.


Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. I. Coupling of alpha 2-adrenergic receptor subtypes to distinct G-proteins. alpha 2-Adrenergic receptor (alpha 2-AR) subtypes couple to pertussis toxin (PT)-sensitive G-proteins to elicit both stimulatory and inhibitory cell responses. Signal specificity may be generated by the ability of the receptor subtypes to "recognize" distinct G-proteins with different affinity. To address this issue we stably expressed three alpha 2-AR subtypes, RNG alpha 2 (alpha 2B-AR), RG10 (alpha 2C-AR), and RG20 (alpha 2D-AR), in NIH-3T3 fibroblasts, which express two PT-sensitive G-proteins (Gi alpha 2, Gi alpha 3), and analyzed receptor/G-protein interactions by determining: 1) functional coupling to adenylylcyclase and 2) the ability of the receptors to exist in a high affinity state for agonist. In alpha 2D-AR transfectants expressing 200 or 2,200 fmol of receptor/mg of protein, epinephrine (10 microM) inhibited forskolin-induced elevation of cellular cAMP by 26 +/- 4.8% and 72 +/- 6.2%, respectively. Similar results were obtained in alpha 2B-AR transfectants. However, in alpha 2C-AR transfectants (200 fmol/mg) the forskolin-induced elevation of cellular cAMP was not altered by agonist treatment. In alpha 2C-AR transfectants expressing higher receptor densities (650-1,200 fmol/mg), epinephrine inhibited the effect of forskolin by 30 +/- 3.2%. This difference in functional coupling among the alpha 2-AR subtypes is reflected at the receptor/G-protein interface. In membrane preparations of alpha 2B and alpha 2D-AR but not alpha 2C-AR transfectants, agonist competition curves were biphasic, indicating high and low affinity states of the receptor for agonist. The high affinity state was guanyl-5'-yl imidodiphosphate- and PT-sensitive, indicative of receptor/G-protein coupling. These data suggest that the alpha 2C-AR differs from the alpha 2B and alpha 2D-AR subtypes in its ability to recognize PT-sensitive G-proteins expressed in NIH-3T3 fibroblasts. The alpha 2C-AR may couple preferentially to PT-sensitive G-proteins (Gi1, Go1,2) not expressed in NIH-3T3 fibroblasts and thereby elicit different cellular responses.


Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. II. Preferential coupling of the alpha 2C-adrenergic receptor to the guanine nucleotide-binding protein, Go. Cell to cell communication by many hormones and neurotransmitters involves three major entities: receptor (R), G-protein (G), and effector molecule (E). Plasticity in this system is conferred by the existence of each entity as isoforms or closely related subtypes that are expressed in a tissue-specific and developmentally regulated manner. Factors that determine signal specificity in this system are poorly understood. Such factors include the relative affinity and stoichiometry of R-G or G-E and the possible colocalization of R-G-E in cellular microdomains. Utilizing the alpha 2-adrenergic receptor (alpha 2-AR) system as a representative subfamily of this class of signal transducers, we determined the relative importance of these factors. By analysis of R-G coupling in mammalian cells cotransfected with alpha 2-AR genes and G alpha cDNA, we demonstrate preferential coupling between an alpha 2-AR subtype and Go. Our data implicate R-G affinity as an important determinant of signal transduction specificity and indicate that a critical level of Go alpha is required for coupling. This report indicates the utility of R-G cotransfection in mammalian cells as a "natural environment model" to characterize events occurring at the R-G and G-E interface.


Accumulation of a microtubule-binding protein, pp170, at desmosomal plaques. The establishment of epithelial cell polarity correlates with the formation of specialized cell-cell junctions and striking changes in the organization of microtubules. A significant fraction of the microtubules in MDCK cells become stabilized, noncentrosomally organized, and arranged in longitudinal bundles in the apical-basal axis. This correlation suggests a functional link between cell-cell junction formation and control of microtubule organization. We have followed the distribution of pp170, a recently described microtubule-binding protein, during establishment of epithelial cell polarity. This protein shows the typical patchy distribution along microtubules in subconfluent fibroblasts and epithelial cells, often associated with the peripheral ends of a subpopulation of microtubules. In contrast to its localization in confluent fibroblasts (A72) and HeLa cells, however, pp170 accumulates in patches delineating the regions of cell-cell contacts in confluent polarizing epithelial cells (MDCK and Caco-2). Double immunolocalization with antibodies specific for cell-cell junction proteins, confocal microscopy, and immunoelectron microscopy on polarized MDCK cells suggest that pp170 accumulates at desmosomal plaques. Furthermore, microtubules and desmosomes are found in close contact. Maintenance of the desmosomal association of pp170 is dependent on intact microtubules in 3-d-old, but not in 1-d-old MDCK cell cultures. This suggests a regulated interaction between microtubules and desmosomes and a role for pp170 in the control of changes in the properties of microtubules induced by epithelial cell-cell junction formation.


Hypertension in the elderly.

Hypertension in the elderly. More than 50% of the U.S. population 65 years and older have elevated systolic pressure with or without elevated diastolic pressure--and thus in either case are at risk for cerebrovascular and cardiovascular sequelae. As therapy lowers diastolic pressure, however, mortality can increase, presumably when blood pressure is brought down to levels that compromise perfusion.


Expression of SV-40 T antigen in the small intestinal epithelium of transgenic mice results in proliferative changes in the crypt and reentry of villus-associated enterocytes into the cell cycle but has no apparent effect on cellular differentiation programs and does not cause neoplastic transformation. The mouse intestinal epithelium represents a unique mammalian system for examining the relationship between cell division, commitment, and differentiation. Proliferation and differentiation are rapid, perpetual, and spatially well-organized processes that occur along the crypt-to-villus axis and involve clearly defined cell lineages derived from a common multipotent stem cell located near the base of each crypt. Nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene were used to establish three pedigrees of transgenic mice that expressed SV-40 large T antigen (TAg) in epithelial cells situated in the uppermost portion of small intestinal crypts and in already committed, differentiating enterocytes as they exited these crypts and migrated up the villus. T antigen production was associated with increases in crypt cell proliferation but had no apparent effect on commitment to differentiate along enterocytic, enteroendocrine, or Paneth cell lineages. Single- and multilabel-immunocytochemical studies plus RNA blot hybridization analyses suggested that the differentiation programs of these lineages were similar in transgenic mice and their normal littermates. This included enterocytes which, based on the pattern of [3H]thymidine and 5-bromo-2'-deoxyuridine labeling and proliferating nuclear antigen expression, had reentered the cell cycle during their migration up the villus. The state of cellular differentiation and/or TAg production appeared to affect the nature of the cell cycle; analysis of the ratio of S-phase to M-phase cells (collected by metaphase arrest with vincristine) and of the intensities of labeling of nuclei by [3H]thymidine indicated that the duration of S phase was longer in differentiating, villus-associated enterocytes than in the less well-differentiated crypt epithelial cell population and that there may be a block at the G2/M boundary. Sustained increases in crypt and villus epithelial cell proliferation over a 9-mo period were not associated with the development of gut neoplasms--suggesting that tumorigenesis in the intestine may require that the initiated cell have many of the properties of the gut stem cell including functional anchorage.


Isolation and purification of human endometrial stromal and glandular cells using immunomagnetic microspheres. Isolation of pure preparations of the different cell populations of human endometrium is a prerequisite for studies of in-vitro function. Sieving of dispersed endometrial cells, followed by adsorption onto immunomagnetic microspheres coated with antibody to Thy-1 was used to separate glandular and stromal cells. The purity of these cell populations was checked with antibodies to cytokeratin and Thy-1. The stromal cells were 98% pure and 90% viable, gland cells were 82% pure with 76% viability. The purified cells were able to proliferate in vitro as shown by thymidine incorporation.


TNF-alpha suppresses CR3-mediated myelin removal by macrophages. Mononuclear cells of the monocyte/macrophage system play an important role in myelin ingestion during Wallerian degeneration. The present in vitro study clarifies the role in this process of two macrophage-secreted cytokines, TNF-alpha and interleukin-1. Treatment with TNF-alpha massively reduced the amount of myelin ingested by macrophages via their complement receptor type 3 (CR3). Anti-TNF-alpha antibodies reversed the effect. Immunofluorescence of macrophages indicated that TNF-alpha caused a reduced expression of the CR3 by phagocytic cells. Further experiments revealed an interaction of TNF-alpha with its receptor on the macrophage cell membrane. Interleukin-1 had no effect on myelin ingestion in the in vitro system used in these experiments.


Restriction endonuclease analysis of Aspergillus fumigatus DNA. To develop a genome based DNA fingerprinting system for Aspergillus fumigatus mould. DNA was extracted from 21 isolates obtained from eight patients with an aspergilloma. This was with a freeze-dried mycelial extract fragmented in liquid nitrogen. DNA was subsequently purified by phenol-chloroform extraction followed by ultracentrifugation on a caesium chloride gradient. The DNA was restricted by EcoRI and Xba I. All isolates were identical when cut by EcoRI; Xba I delineated six DNA types. DNA fingerprinting can be used to type isolates of A fumigatus. Strains from within an aspergilloma which were morphologically distinct could either have the identical DNA fingerprint or produce a unique type.


Pharmacotherapy in the prevention of suicidal behavior. Prevention or reduction of suicide remains a serious challenge for the medical community. Psychotherapeutic and psychosocial interventions have not been shown to reduce the incidence of suicide attempts and analytic psychotherapy has been shown to increase suicidal behavior. The efficacy of pharmacotherapy in reducing suicide attempts in patients with a history of repeated suicidal behavior has been shown with low doses of the neuroleptic agent flupenthixol compared with placebo. Supporting findings have been reported with trifluoperazine. There is evidence to suggest that some antidepressants may not be neutral in their effect on suicidal behavior. Maprotiline, for example, was associated with an increase in suicide attempts compared with placebo in a large long-term treatment study despite its significant efficacy in preventing relapse of depression. Differential lethality indices taken from large community studies support the notion that noradrenergic drugs such as maprotiline, desipramine, and nortriptyline are associated with a higher than expected incidence of death from overdose, and the suicide-provoking potential may relate to some noradrenergic property. The studies of serotonergic antidepressants do not suggest that they are suicide-provoking agents; rather they appear to be neutral or protective. Prospective prophylactic studies are needed to test the ability of potential treatments for the reduction of suicidal behavior.


Projections from rabbit caudal medulla to C1 and A5 sympathetic premotor neurons, demonstrated with phaseolus leucoagglutinin and herpes simplex virus. We combined Phaseolus vulgaris leucoagglutinin anterograde tracing and Herpes simplex virus transneuronal retrograde tracing to determine whether neurons in the vasodepressor region of the rabbit caudal ventrolateral medulla project to brainstem neurons containing the virus after its transneuronal transport from the adrenal medulla. Five days after adrenal injection of virus, 764 +/- 159 virus-positive neurons were found bilaterally in the brainstem: 61% in the C1 sympathoexcitatory region of the rostral ventrolateral medulla, 30% in the A5 region, 5% in the parapyramidal region, and 3% in the paraventricular nucleus of the hypothalamus. Many of the virus-positive neurons in the C1 and A5 areas also contained tyrosine hydroxylase and, in the parapyramidal area, many contained 5-hydroxytryptamine. After iontophoretic deposit of leucoagglutinin into the vasodepressor region of the caudal ventrolateral medulla, brain regions containing varicose processes labeled with leucoagglutinin included the regions containing virus-positive neurons. We examined the C1 and A5 regions following injections of both tracers in the same rabbits, leucoagglutinin into the caudal ventrolateral medulla and virus into the adrenal gland. Varicosities containing leucoagglutinin were seen in contiguity with perikarya and dendritic branches of neurons containing HSV1, in both the C1 and A5 regions. Studies also revealed labeled varicosities in contiguity with TH-containing C1 and A5 neurons. The projection from the caudal medulla to presumed sympathetic premotor neurons in the C1 area, including some C1 cells, represents a potential pathway whereby activity of neurons in the caudal medulla could reduce blood pressure by inhibiting sympathoexcitatory neurons in the rostral medulla.


Activation of human phagocytes through carbohydrate antigens (CD15, sialyl-CD15, CDw17, and CDw65). The leukocyte carbohydrate (CHO) Ag CD15, sialyl-CD15, and CDw65 have recently been found to function as ligands for CD62 and ELAM-1 cell adhesion molecules on platelets and endothelium, respectively. Cell adhesion ligands also may act as receptors capable of signal transduction. We therefore investigated the possibility that these CHO Ag and CDw17, a glycolipid Ag whose expression is regulated by leukocyte activation, may have receptor-like characteristics. The effects of antibody cross-linking of CHO Ag on phagocyte activation were measured by using flow cytometry and fluorescent indicators for cytoplasmic calcium ions, oxidative burst, and the granule-associated proteins CD11b and CD67. Cross-linking of CD15, sialyl-CD15, CDw65, or CDw17 induced a moderate release of calcium ions into the cytoplasm of granulocytes, a strong activation of oxidative burst, and a low up-regulation of CD11b and CD67 compared to the effects of treatment with 4 microM FMLP. The results suggest a role for CHO Ag in leukocyte signal transduction and support the view that these molecules are involved in phagocyte activation.


Internalization of a major group human rhinovirus does not require cytoplasmic or transmembrane domains of ICAM-1. Intercellular adhesion molecule-1 (CD54), a cell adhesion molecule and the receptor for the major group of rhinoviruses, is a class 1 membrane protein with five Ig-like domains in its extracellular region, a transmembrane domain, and a short cytoplasmic domain. The amino-terminal domains (D1 and D2) are sufficient for virus binding and the first is most important (1). We have investigated whether other extracellular domains, transmembrane or cytoplasmic domains are required for virus entry as determined by postinfection virion protein biosynthesis. We demonstrate that cytoplasmic, transmembrane, and Ig-like domains 3, 4, and 5 are not essential for rhinovirus entry into transfected COS cells. The efficiency of rhinovirus infection directly correlates with the efficiency of rhinovirus binding and a form of intercellular adhesion molecule-1 that is glycophosphatidyl-inositol anchored, and thus does not extend into the inner leaflet of the membrane bilayer or the cytoplasm efficiently supports virus entry.


In vivo protection by the aminothiol WR-2721 against neutron-induced carcinogenesis. The ability of the compound S-2-(aminopropylamino)ethylphosphorothioic acid, designated WR-2721, to protect against neutron-induced carcinogenesis was investigated. Both sexes of the B6CF1 mouse were injected i.p. with 400 mg/kg of WR-2721 30 min prior to being irradiated by 10 cGy of neutrons. Neoplastic mortality in the groups receiving thiol was either reduced or delayed relative to irradiated mice not given protector. However, the time at which the protective effect of WR-2721 was expressed depended on the sex of the animal. Thiol-related shifts in the time of neoplastic death in females occurred only in the first half of the lifespan. Once a female survived to the mean age at death, no difference in the pattern of mortality could be detected between control and WR-2721-treated mice exposed to neutrons. Irrespective of thiol treatment, the timing of tumour-related death was nearly identical during the first half of life for males exposed to neutrons. In the last half of the lifespan, survival of thiol-protected males was enhanced relative to saline-injected males and even exceeded that observed in the control population.


Reaction of dithiothreitol and para-nitroacetophenone with different radical precursors of .OH radical-induced strand break formation of single-stranded DNA in anoxic aqueous solution. The yields of single-strand breakage (ssb) in single-stranded calf thymus DNA (ssDNA) have been determined after 60Co gamma-irradiation of aqueous anoxic solutions in the presence of different concentrations of dithiothreitol (DTT), ascorbate or trans-4,5-dihydroxy-1,2-dithiane, using low-angle laser light scattering. The influence of DTT on the kinetics of ssb formation has been determined by conductivity measurements in pulse radiolysis. The results suggest that strand breakage in ssDNA proceeds via two modes of about equal contribution and with half-lives of about 7 ms and 0.8s, respectively. Both modes reflect reactions of at least two DNA radicals, which react with DTT by hydrogen-atom transfer reactions with similar rate constants of about 5-9 x 10(5) dm3 mol-1 s-1. These hydrogen-atom transfer reactions inhibit strand break formation. The slow mode is shown to represent the decay of base-radicals to generate sugar radicals. The involvement of the oxidizing .OH adduct radical of guanine in the formation of strand breaks can be ruled out and there is no evidence for a contribution from the anion or radical anion of DTT to the inhibition of strand breaks via electron transfer reactions to DNA radicals.


Catalytic reduction of Fe(III)-cytochrome-c involving stable radiolysis products derived from disulphides, proteins and thiols. gamma-Radiolysis, with doses less than 1 kGy, of aqueous solutions of disulphides, disulphide-proteins or thiols leads to the generation of stable products, capable of stimulating the catalytic reduction of Fe(III)-cytochrome-c by unirradiated glutathione, and by other thiols. The stimulatory activity fades within 20-60 min in the case of irradiated thiols, but there was little loss of this activity when irradiated solutions of disulphides or disulphide-proteins were stored at 4 degrees C for days. Disulphides (e.g. cystamine) are mainly activated by .OH radicals, disulphide-proteins (e.g. alpha-chymotrypsin) mainly by e-aq, and thiols (e.g. cysteine) by virtually all water radicals. The radiolytic activation, which is only partially prevented by oxygen, can be attributed to the generation of trace amounts of higher sulphides and persulphides (RSSSR, RSSSSR and RSSH). Such species are known to stimulate Fe(III)-cytochrome-c reduction by glutathione in a chain reaction (Massey et al. 1971). The radiolytic stimulation of reductive catalytic activity of thiols and disulphides may play a role in irradiated biological systems, and might be exploited to identify irradiated proteins with Fe(III)-cytochrome-c as detector.


Substituent effects in the free radical reactions of silybin: radiation-induced oxidation of the flavonoid at neutral pH. Silybin dihemisuccinate sodium salt, a flavonoid used in human therapy of liver dysfunction, has an inhibitory effect in vivo on radiation-induced deactivation of enzymes and peroxidation of membrane lipids in rat liver microsomes. The reactivity of silybin and its phenolic OH groups towards free radicals (OH, N3., (SCN)2.-, Cl3CO2.) in aqueous solution was studied by pulse radiolysis. Absorption spectra for the phenoxyl-type radicals were assigned using structurally similar models. The one-electron reduction potential for silybin at pH 7 (E07 = 0.76 V), determined using the p-methoxy-phenoxyl/phenolate redox couple as reference standard (E07 = 0.72 V, Lind et al. 1990), is related to the 3'-methoxy-4'-OH structure, the exclusive target for one-electron oxidation at pH 7, while the 7-OH and 5-OH groups are prevented from oxidation by 4-keto substitution and intramolecular H-bonding, respectively. The free radical reactivity of silybin compares favourably with poly-OH-substituted flavonoids; however, the latter compounds have been reported to generate potentially toxic oxygen species at a biologically relevant pH.


Direct comparison between protons and alpha-particles of the same LET: I. Irradiation methods and inactivation of asynchronous V79, HeLa and C3H 10T1/2 cells. A direct comparison was carried out of the biological effectiveness of protons and alpha-particles of the same linear energy transfer (LET) under identical conditions with a variety of in vitro biological systems. Monolayers of mammalian cells were irradiated with accelerated beams of protons (1.2 and 1.4 MeV) and alpha-particles (30 and 35 MeV) corresponding to LETs of 23 and 20 keV microns-1 for each particle type. For V79-4 cells it was observed that the linear term of the dose-response for cell inactivation by protons was significantly greater than that for alpha-particles of the same LET. For HeLa and HeLa S3 cells, also, the linear term appeared to be greater for protons, but this was not observed with more limited data for C3H 10T1/2 cells. The result for V79 cells is in agreement with the report of Belli et al. (1989) who observed that the biological effectiveness of protons rose sharply between 17 and 30 keV microns-1 in strong contrast to alpha-particles which reached a peak effectiveness at greater than 100 keV microns-1. These results place new constraints on the biologically relevant features of the microscopic structure of radiation tracks, and have implications for the mechanistic and practical comparison between radiations.


Direct comparison of biological effectiveness of protons and alpha-particles of the same LET. II. Mutation induction at the HPRT locus in V79 cells. Mutation induction at the hprt locus has been studied in V79-4 Chinese hamster cells irradiated with mono-energetic protons and alpha-particles with LET of 20.3 and 23 keV microns-1. The mutation frequency increased linearly with the dose for all the four radiation qualities investigated, so that effectiveness for mutation induction could be expressed by the slope of the relevant curve. This effectiveness did not significantly change with the small change in LET of each kind of particle, while sizeable differences were found between particles. Protons were more effective in mutation induction than alpha-particles with the same LET by a factor of about 2. This finding is similar to, although slightly larger than, the factors 1.5-1.8 found for inactivation of the same cells in the same series of experiments.


Direct comparison of biological effectiveness of protons and alpha-particles of the same LET. III. Initial yield of DNA double-strand breaks in V79 cells. The results reported form part of a series of experiments to substantiate and extend the findings by Belli et al. (1989) that protons are more biologically effective at cell killing than alpha-particles of the same LET. The irradiations were carried out using the Variable Energy Cyclotron (VEC) at the Harwell Laboratories. V79-4 Chinese hamster cells were exposed to alpha-particles and protons with LETs of 20 and 23 keV microns-1 in the dose range 40-150 Gy. X-rays were also used for comparison. Two methods were used for measurement of initial DNA double-strand breaks: sedimentation and DNA precipitation assays. The dose-response relationships were found to be well fitted by straight lines in all cases. With the sedimentation assay a slightly lower yield of dsb was found from protons than from alpha-particles of the same LET. The yield from X-rays was not significantly different from either. The precipitation assay showed similar yields of DNA damage from both particle types but significantly higher yields from X-rays. This may reflect a difference in the type of lesions scored by the two methods. Since the initial amount of dsb does not account for the observed differences in cellular response to radiations of different qualities, it is likely that these are related to the nature of the dsb (affecting reparability) or to the occurrence of other types of molecular damage.


Kinetics of micronucleus induction by 125I-labelled thyroid hormone in hormone-responsive cells. Two cell lines, CHO and GC, different in their tissue origin, were investigated with the aim of discovering the correlation between the level of 125I-T3 binding and chromosomal damage induced by 125I decay. Incubation of cells with 125I-T3 has been performed in two exposure schedules: continuous incubation for one to six cell cycles and a pulse-chase schedule involving exposure for one cell cycle. The cellular uptake of 125I-T3, its compartmentization and kinetics were different in the two cell lines. GC cells contained about 7 times more 125I-T3 than CHO cells when incubated with the same external 125I activity concentration (74 kBq of 125I-T3 ml-1 medium). Approximately 70% of the cellular 125I-T3 was found in nuclei of GC cells and only 5% in the nuclei of CHO cells. During the long-term incubation of GC cells with 74 kBq of 125I-T3 ml-1 medium, the 125I activity concentration in cells and their nuclei initially decreased by a half, and thereafter reached a plateau after the third doubling time. In CHO cells and nuclei a very slow linear increase of 125I activity was observed. In GC cells, micronucleus frequency was found to be correlated with nuclear 125I activity. One cell cycle pulse labelling with 74 kBq of 125I-T3 ml-1 medium caused a significant enhancement of micronucleus frequency above the control level during six doubling times, with a maximum at the first post-labelling doubling time. In GC cells continuously incubated with 74 kBq of 125I-T3 ml-1 medium, the micronucleus frequency increased with the incubation time. A model of T3 receptor-dependent dose delivery to nuclei of GC cells continuously incubated with 125I-T3 is proposed. The frequency of micronuclei in the CHO cell line continuously incubated with 125I-T3 did not differ significantly from the control, whereas in the pulse-chase schedule the mean frequency of micronucleated binuclear cells was lower during 4 post-labelling doubling times (significantly at the first and second post-labelling doubling time and insignificantly at the later doubling times) than in the control. Incubation of GC cells with various activity concentrations in medium for four cell cycles resulted in a linear increase of 125I activity in cells and nuclei; however, with a saturation in the region of highest 125I-T3 concentrations used. The frequency of binuclear cells bearing micronuclei was linearly dependent on the nuclear 125I-T3 concentration.


Intra- and inter-individual variation of background and radiation-induced micronucleus frequencies in human lymphocytes. Serial blood samples were taken from four healthy individuals (three males, one female, aged between 26 and 51 years) in 3-monthly intervals during 1 year. Leucocyte suspensions were prepared and exposed to 3 Gy of 137Cs gamma-rays or left unirradiated as controls. In a cytokinesis-blocked (CB) micronucleus (MN) assay significant inter- and intra-donor variations of background and radiation-induced MN incidences became apparent. The two sources of variation lead to an extra variance sigma I2, in addition to the sample variance sigma e2 of MN incidences. The contributions of the different components to the total variance were estimated by means of a variance component model. The deviation sigma I for the mean background MN level of 1.53 x 10(-2) MN/CB cell was +/- 0.67 x 10(-2) and for the mean radiation-induced MN level of 0.53 MN/CB cell it was +/- 0.10. The contribution of the intra-individual variance to sigma I2 was about 50% for background MN levels and 75% for radiation-induced MN frequencies. With respect to the application of the CB-MN assay as a biological dosimetry system, the consequences of the present findings for calibration purposes and low-dose estimation are discussed. The calculation of the variance components is explained in an appendix, which serves also as an example for the adaptation of analysis of variance techniques to the evaluation of data derived from scoring of MN, as well as from scoring of metaphase chromosomal aberrations.


Role of protein kinase-C in thymocyte apoptosis induced by irradiation. The role of protein kinase C in radiation-induced death of thymocytes was studied. For this purpose murine thymocytes were irradiated and incubated for 6 h at 37 degrees C and afterwards the fraction of fragmented DNA was measured. Results indicate that radiation-induced DNA fragmentation can be prevented by adding the protein kinase C inhibitor H-7 or staurosporine to the thymocytes during incubation time. Incubation of irradiated cells with HA-1004, an inhibitor of cAMP-dependent protein kinase, with a minor effect on protein kinase C did not affect the DNA fragmentation induced by irradiation. Incubation of cells with phorboldibutyrate gave a dose-dependent induction of DNA fragmentation. This effect can be inhibited by staurosporine. These results suggest that radiation-induced DNA fragmentation is an active cellular process in which protein kinase C plays an important role.


Effects of cycloheximide and actinomycin D on radiation-induced apoptotic cell death in the developing mouse cerebellum. Effects of cycloheximide and actinomycin D on radiation-induced cell death in the external granular layer (EGL) of the cerebellum were studied in vivo. Newborn mice were exposed to 0.24 Gy gamma-radiation, and dying cells which exhibited pyknosis of nuclei in the EGL were examined at various post-irradiation periods. The number of pyknotic cells began to increase 3 h after irradiation, reached a peak incidence at 6 h, and then gradually fell to the sham-irradiated level by 18 h. When pups were injected with cycloheximide 1 h after irradiation, cell death was suppressed for 6 h, but a peak mortality as high as in the case of radiation alone was attained at 15 h after irradiation. When pups were treated with cycloheximide twice, at 1 and 6 h after irradiation, cell death did not occur for 15 h, but then the incidence rose to a level similar to that after irradiation alone. These findings showed that radiation-induced cell death in the EGL is suppressed by cycloheximide until the chemical is metabolized. Hence, death is by apoptosis which is known to require macromolecular synthesis, and the 'signal' for apoptosis in the cell persists for at least 15 h after irradiation. On the other hand, actinomycin D injected immediately before or after irradiation did not affect the initiation of cell death; actinomycin D alone induced cell death.


High radiosensitivity of the mineralization capacity of adult murine bone marrow in vitro to continuous alpha-irradiation compared to acute X-irradiation. Adult BALB/c mice, injected with osteosarcomogenic amounts of 241Am (between 40 and 500 Bq/g mouse) showed an impaired mineralization capacity of their femoral bone marrow. This effect persisted until at least 1 year after 241Am injection and was expressed after incubation of bone marrow cells in vitro in conditions allowing osteogenic differentiation. The mineralization capacity of marrow in vitro was evaluated by measurement of 85Sr uptake from the tissue culture medium. Two osteogenic assays were used: in marrow cultured as an intact organ (marrow organ cultures), reduced mineralization was observed in mice given 149 Bq 241Am/g mouse or more (skeletal dose rate of 25 mGy/day), in stromal marrow cells cultured from adherent cell layers and subsequently brought into a three-dimensional (3D) mineralizing condition (stromal 3D cultures), reduced 85Sr uptake was observed from the lowest dose level tested (42 Bq 241Am/g mouse, skeletal dose rate of 7 mGy/day). Taking into account that only a fraction of the skeletal alpha-dose reached the marrow of the femoral diaphyses, marrow organ cultures and stromal 3D cultures exhibited high radiosensitivity to alpha-irradiation in vivo. However, after acute X-irradiation of marrow in vivo or in vitro prior to initiation of the marrow organ cultures, X-ray doses of 4 Gy or higher were needed to significantly impair the mineralization capacity of marrow organ cultures in vitro. Our data demonstrated that the osteogenic cells from the bone marrow are subjected to long-term damage after low doses of continuous alpha-irradiation in vivo.


Accumulation of glycosaminoglycans in radiation-induced muscular fibrosis. The content and biosynthesis of glycosaminoglycans (GAGs) were studied in the pig thigh muscle after acute local gamma-irradiation. Seven months following irradiation, the muscular tissue next to the irradiation cone was replaced by severe mutilating fibrosis delimited by an intermediary perifbrotic zone. Fibrosis, perifibrotic tissue and normal muscle, were sampled and incubated with [3H]glucosamine and [35S]sulphate, and GAGs were isolated following pronase digestion. Results showed a parallel increase of collagen and GAG content in perifibrotic and fibrotic tissues. Sulphated GAGs, heparan sulphate and dermatan sulphate were preferentially accumulated in fibrotic tissue, while the hyaluronic acid content increased only slightly. Synthesis of sulphated GAGs was more elevated in fibrotic tissue than in perifibrotic zone as compared with normal muscle. Seven months after irradiation well-developed fibrotic tissue continued to synthesize and to accumulate extracellular matrix macromolecules, indicating the invasive aspect of post-irradiation fibrosis.


Radioprotective effect of exogenous glutathione on rat parotid glands. The prophylactic effect of glutathione (GSH) on radiation injury in rat parotid glands was investigated. GSH was administered to male Wistar rats i.p. 15 min prior to irradiation. The necrosis index (NI) of the glands was determined histologically 24 h after a single dose of 15, 30, or 60 Gy. Total amylase activity, total protein content and wet weight of the glands were measured 30, 60 and 90 days after irradiation. Administration of GSH prior to radiation minimized acute and chronic radiation injuries as a function of the GSH dose: i.e. reduction of NI and prevention of the decrease in total amylase activity, total protein content and gland weight. The intraglandular level of non-protein-bound thiols (NPSH) and GSH increased significantly after i.p. administration of GSH, whether or not the glands were irradiated. An autoradiographic study revealed that i.p.-administered 35S-GSH was actively taken up by the glandular parenchyma, especially in the acini and ducts. It was shown that elevation of the intraglandular level of NPSH after exogenous administration of GSH protected the parotid glands from radiation injury in the rat.


Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation. The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)


Excitatory postsynaptic potentials recorded from regular-spiking cells in layers II/III of rat sensorimotor cortex. 1. Intracellular recording techniques were used to investigate the physiological and pharmacological properties of stimulus-induced excitatory postsynaptic potentials (EPSPs) recorded in regular-spiking cells located in layers II/III of rat sensorimotor cortical slices maintained in vitro. 2. Depending on the strength of the extracellular stimuli, a pure EPSP or an EPSP-inhibitory postsynaptic potential sequence was observed under perfusion with normal medium. The EPSPs displayed short latency of onset [2.4 +/- 0.7 (SD) ms] and were able to follow repetitive stimulation (tested less than or equal to 5 Hz). Variation of the membrane potential (Vm) revealed two types of voltage behavior for the short-latency EPSP. The first type decreased in amplitude with depolarization and increased in amplitude with hyperpolarization. In contrast, the second type behaved anomalously by increasing and decreasing in size after depolarization and hyperpolarization, respectively. 3. Several experimental procedures were carried out to investigate the mechanism underlying the anomalous voltage behavior of the EPSP. Results indicated that this type of Vm dependency could be mimicked by an intrinsic response evoked by a brief pulse of depolarizing current and could be abolished by N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (50 mM). Furthermore, the EPSP was not sensitive to the N-methyl-D-aspartate (NMDA) receptor antagonist 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP, 10 microM). Thus the anomalous voltage relationship of the neuronal membrane. 4. The involvement of non-NMDA receptors in excitatory synaptic transmission was investigated with their selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 1-10 microM). This drug greatly reduced or completely blocked the EPSP in a dose-dependent manner (1-10 microM). The IC50 for the CNQX effect was approximately 2 microM. In the presence of CNQX (10 microM) and glycine (10 microM), synaptic stimulation failed to elicit firing of action potential. However, a CPP-sensitive EPSP was observed. 5. When synaptic inhibition was reduced by low concentration of bicuculline methiodide (BMI, 1-2 microM), extracellular stimulation revealed late EPSPs (latency to onset: 10-30 ms) that were not discernible in normal medium. Similar to the short-latency EPSP, the Vm dependency displayed by this late EPSP could be modified by inward membrane rectifications. The late EPSP appeared to be polysynaptic in origin because 1) its latency of onset was long and variable and 2) it failed to follow repetitive stimuli delivered at a frequency that did not depress the short-latency EPSP.(ABSTRACT TRUNCATED AT 400 WORDS)


Identified FMRFamide-immunoreactive neuron LPL16 in the left pleural ganglion of Aplysia produces presynaptic inhibition of siphon sensory neurons. The gill- and siphon-withdrawal reflex of Aplysia undergoes transient inhibition following noxious stimuli such as tail shock. This behavioral inhibition appears to be due in part to transient presynaptic inhibition of the siphon sensory cells, which can be mimicked by application of the peptide FMRFamide. Although FMRFamide is widespread in the Aplysia nervous system, an FMRFamide-containing inhibitory neuron has not previously been identified. We have searched for such a neuron by combining FMRFamide immunofluorescence with fluorescent dye backfilling from the abdominal ganglion, the location of the siphon sensory cells. These methods localized a neuron in the left pleural ganglion, which we have named LPL16. LPL16 is FMRFamide immunoreactive; it is excited by tail shock; and stimulation of LPL16 produces inhibition of siphon sensory cell-to-motor cell postsynaptic potentials and narrowing of action potentials in the sensory cells in tetraethylammonium solution. These results indicate that LPL16 participates in the inhibitory effects of tail shock, and support the idea that FMRFamide plays a physiological role in the inhibition.


Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons. Glutamate-induced changes in intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single rat hippocampal neurons grown in primary culture by employing the Ca2+ indicator indo-1 and a dual-emission microfluorimeter. The [Ca2+]i was monitored in neurons exposed to 100 microM glutamate for 5 min and for an ensuing 3 hr period. Ninety-two percent (n = 64) of these neurons buffered the glutamate-induced Ca2+ load back to basal levels after removal of the agonist; thus, the majority of cells had not lost the ability to regulate [Ca2+]i at this time. However, following a variable delay, in 44% (n = 26) of the neurons that buffered glutamate-induced Ca2+ loads to basal levels, [Ca2+]i rose again to a sustained plateau and failed to recover. The changes in [Ca2+]i that occur during glutamate-induced delayed neuronal death can be divided into three phases: (1) a triggering phase during which the neuron is exposed to glutamate and the [Ca2+]i increases to micromolar levels, followed by (2) a latent phase during which the [Ca2+]i recovers to a basal level, and (3) a final phase that begins with a gradual rise in the [Ca2+]i that reaches a sustained plateau from which the neuron does not recover. This delayed Ca2+ overload phase correlated significantly with cell death. The same sequence of events was also observed in recordings from neuronal processes. The delayed Ca2+ increase and subsequent death were dependent upon the presence of extracellular Ca2+ during glutamate exposure. Calcium influx during the triggering phase resulted from the activation of both NMDA and non-NMDA receptors as indicated by studies using receptor antagonists and ion substitution. Treatment with TTX (1 microM) or removal of extracellular Ca2+ for a 30 min window following agonist exposure failed to prevent the delayed Ca2+ overload. The delayed [Ca2+]i increase could be reversed by removing extracellular Ca2+, indicating that it resulted from Ca2+ influx. The three phases defined by changes in the [Ca2+]i during glutamate-induced neuronal toxicity suggest three distinct targets to which neuroprotective agents may be directed.


Direct in vitro electrochemical measurement of dopamine overflow in intermediate pituitary: characterization and pharmacology. The intermediate lobe of the pituitary (IL) is a promising model system for the study of dopaminergic neurotransmission. We sought to characterize the release of dopamine (DA) from the IL using electrochemical methods which provide increased spatial and temporal resolution compared to the superfusion techniques which had previously been employed to study this system. After pretreatment with monoamine oxidase (MAO) inhibitors, we were able to routinely measure DA overflow after 30-sec, 9-Hz electrical stimulation of the pituitary stalk. This effect was blocked by tetrodotoxin and Cd++ ions confirming that it was due to synaptic activation. Intracellular recording from the postsynaptic cells (melanotrophs) showed a postsynaptic hyperpolarization with a time course that was identical to that of DA overflow. Overflow was increased and prolonged by agents that inhibited DA uptake (nomifensine, mazindol or cocaine) and increased but not prolonged by a D2 antagonist (sulpiride), presumably acting via presynaptic autoreceptors. The DA precursor 3,4-dihydroxyphenylalanine also increased DA overflow gradually over a 45-min time period. In view of reports of possible colocalization of DA and gamma-aminobutyric acid in IL, we determined the possible effects of GABAergic agents on DA release. Neither agonists (muscimol or baclofen) nor antagonists (bicuculline or 2-hydroxysaclofen) had any effect on DA overflow. By using the MAOA-specific inhibitor clorgyline and the MAOB-specific inhibitor deprenyl we were able to confirm that the MAOA form of the enzyme is involved in DA metabolism in this preparation.


Effects of 3,4-dihydroxymethamphetamine and 2,4,5-trihydroxymethamphetamine, two metabolites of 3,4-methylenedioxymethamphetamine, on central serotonergic and dopaminergic systems. The effects of 3,4-dihydroxymethamphetamine (DHM) and 2,4,5-trihydroxymethamphetamine (THM) on central serotonergic and dopaminergic systems were investigated to determine if these metabolites share the neurochemical properties of 3,4-methylenedioxymethamphetamine. THM (50-200 micrograms) or DHM (135 micrograms) was administered i.c.v. to rats; 5 days later, cortical, striatal and hippocampal tryptophan hydroxylase (TPH) activity were decreased by THM in a dose-dependent manner, whereas DHM was without effect in these brain structures. The concentration of serotonin in the brain structures contralateral to the side of THM injection was also decreased, but to a lesser degree. THM (100 and 200 micrograms) increased TPH activity to 155% of control in the dorsal raphe, whereas a dose of 50 micrograms increased TPH activity to 132% of control in the median raphe nucleus. THM also markedly reduced striatal tyrosine hydroxylase activity, but did not alter enzyme activity in the substantia nigra; DHM increased striatal tyrosine hydroxylase activity to 115% of control. These results suggest that THM, but not DHM, is toxic to both dopaminergic and serotonergic nerve terminals. Although THM could not be established as the neurotoxic metabolite explaining 3,4-methylenedioxymethamphetamine (MDMA) toxicity, its properties may prove useful in elucidating amphetamine toxicity.


Increased rates of punished responding produced by buspirone-like compounds in rats. Buspirone has been reported to have effects on punished responding in rats which are considerably smaller and less reliable than those produced by benzodiazepines. A recent study, however, found that buspirone and ipsapirone increased operant responding suppressed by a stimulus which preceded unavoidable shock, suggesting that conditioned emotional response protocols may be more sensitive than punishment procedures. This hypothesis was not supported by the results of the present study. The food-reinforced lever pressing of rats was maintained by a multiple VI schedule with punished and unpunished components. The experimental parameters were similar to those used in the previous conditioned emotional response study. The benzodiazepines, chlordiazepoxide and clorazepate, as well as buspirone (at one dose) and ipsapirone (at several doses), increased rates of punished responding. The 5-hydroxytryptamine-1A receptor ligands 8-hydroxy-2-(di-n-propylamino)tetralin and MDL 73005EF did not reliably produce similar effects, although increased rates of punished responding were seen in some animals. Haloperidol, imipramine and idazoxan did not increase punished response rates. Although the increases in punished responding produced by buspirone were smaller than those produced by chlordiazepoxide and clorazepate, the effect of ipsapirone was quantitatively similar to that of the benzodiazepines.


Use of 2-hydroxypropyl-beta-cyclodextrin as an intrathecal drug vehicle with opioids. 2-Hydroxypropyl-beta-cyclodextrin (CDEX), a seven-membered glucose pyranose structure, forms reversible inclusion complexes with the lipophilic portion of a drug molecule by noncovalent bonding. This can increase the water solubility of lipid-soluble drugs and reduce the rate of clearance of such agents from the spinal cord into the vasculature after i.t. administration. In this study, opioids (morphine, lofentanil, alfentanil and sufentanil) with and without CDEX (20, 2, 0.2 and 0.02% w/v in sterile water) were administered spinally in rats prepared with chronic i.t. catheters. CDEX prolonged the duration of analgesia (52.5 degrees C hot plate) and reduced the incidence of catalepsy otherwise produced by a supermaximal i.t. dose of each of the opioids. The magnitude of the potentiating effect of CDEX on opioids was dependent upon concentration of the CDEX and varied with drug lipid partition coefficients. The highest concentration of CDEX alone (20%) had no effect upon the volume-evoked micturition reflex, blood pressure, heart rate, or spinal reflexes. Our data indicate that CDEX may be a useful i.t. vehicle for modifying the redistribution characteristics of highly diffusible molecules after their i.t. administration, and that for each drug there is an optimal CDEX concentration. In the present case, CDEX prolongs the spinal analgesic action and reduces the supraspinal actions of i.t. drugs.


Perinatal changes in the coupling of beta 1- and beta 3 adrenergic receptors to brown fat adenylyl cyclase. The stimulation of adenylyl cyclase by catecholamines in neonatal brown adipose tissue (BAT) is markedly biphasic, suggesting the existence of receptors that have both high and low affinities for catecholamines. The identities of these receptors were examined by comparing responses in neonatal BAT membranes to those of Chinese hamster ovary cells which had been transfected to express the cloned rat beta 1 and beta 3 receptors. The results from these experiments indicate that high-affinity stimulation of adenylyl cyclase by catecholamines in BAT is mediated by beta 1 receptors, as evidenced by the potencies of norepinephrine and isoproterenol at this receptor and the potent blockade of the receptor by alprenolol. The low-affinity catecholamine receptor appears to be the beta 3 receptor, as indicated by the low potency of catecholamine agonists and the inability of low concentrations of alprenolol to block this activity. Furthermore, this receptor, like the cloned rat beta 3 receptor, was antagonized by (-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one (CGP 12177) and was stimulated by (R',R')-4-(2-[(2[(3-chlorophenyl)-2- hydroxyethyl]amino)propyl]phenyl)phenoxyacetic acid (BRL 37344). These results indicate that both beta 1 and beta 3 receptors couple to adenylyl cyclase in BAT and that activation of adenylyl cyclase in neonatal BAT is mediated primarily by beta 3 receptors. Beta 3 receptors were also clearly detected in weanling BAT with the beta 3-selective agonist BRL 37344. However, when catecholamines were used to stimulate activity, the activation of adenylyl cyclase by beta 1 receptors, which occurred at low concentrations of catecholamines, obscured the activation of adenylyl cyclase by beta 3 receptors, which occurred only at high concentrations.


Effects of agonist exposure on the coupling of beta 1 and beta 3 adrenergic receptors to adenylyl cyclase in isolated adipocytes. The coupling of beta 1 and beta 3 adrenergic receptors to adenylyl cyclase was examined in membranes of isolated white adipocytes. The activation of adenylyl cyclase by isoproterenol (ISO) was biphasic. The high-affinity activation of adenylyl cyclase, which occurred at submicromolar concentrations of ISO, was mediated by beta 1 receptors, whereas low-affinity activation was mediated by beta 3 receptors. The relative activation of adenylyl cyclase by beta 3 receptors was less when ISO was used to stimulate activity, compared to when the beta 3-selective agonist BRL 37344 was used. These data indicate that both receptor subtypes can stimulate the same adenylyl cyclase in membranes of control cells, and that the activation of adenylyl cyclase by beta 1 receptors by low concentrations of catecholamines can obscure the activation by beta 3 receptors by high concentrations of catecholamines. Exposure of adipocytes to ISO at concentrations that either selectively stimulate beta 1 receptors or nonselectively stimulate both beta receptor subtypes greatly decreased the ability of beta 1, but not beta 3, receptors to activate adenylyl cyclase. In contrast, the exposure of cells to the beta 3-selective agonist BRL only slightly desensitized beta 1 receptors and did not affect beta 3 receptor activation of adenylyl cyclase. These data indicate that acute agonist exposure desensitizes beta 1, but not beta 3, receptors.


Centrally mediated cardiovascular actions of dynorphin A(1-8) on rat hippocampal formation. The effects of dynorphin A(1-8) (DA1-8) microinjected into various areas of the hippocampal formation (HF) on the mean blood pressure (MBP) and heart rate (HR) were investigated in the alpha-chloralose-anesthetized rat. Intra-HF injection of DA1-8 dose-dependently (0.5-50 nmol) reduced MBP and HR. Depressor and bradycardic responses also occurred following microinjection of the excitatory amino acid l-glutamate (1 M, 0.2-0.4 microliter) into the DA1-8-sensitive sites. By contrast, administration of 4% lidocaine (0.2-0.4 microliter) into the same HF sites failed to affect MBP and HR. Pretreatment of the HF with the kappa opioid receptor antagonist nor-binaltorphimine at a dose of 2 to 4 nmol, which itself had no significant influence on basal MBP and HR, almost totally abolished the depressor and bradycardic responses induced by HF injection of DA1-8. DA1-8 at a dose of 10 nmol produced no significant alterations in the frequency of respiration and blood PaO2 and PaCO2 and artificial ventilation did not change the cardiovascular responses of DA1-8. Atropine given i.v. almost totally eliminated the bradycardia and partially prevented the hypotensive responses to intra-HF DA1-8. The data indicate that exogenous administration of DA1-8 into the HF is capable of producing substantial inhibition of peripheral cardiovascular function. Because lidocaine was without effect, the hypotension and bradycardia most likely resulted from an augmentation of an excitatory process rather than from direct inhibition of hippocampal neurons around the injection sites. The effects appear to involve activation of kappa opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatic cytochrome P-450-mediated activation of rat aortic guanylyl cyclase by glyceryl trinitrate. Previous studies have demonstrated hepatic cytochrome P-450-dependent biotransformation of organic nitrates. We assessed whether this biotransformation resulted in the formation of an activator of guanylyl cyclase using the 100,000 x g supernatant of rat aorta as a source of crude enzyme. Incubation of aortic supernatant with rat hepatic microsomes and glyceryl trinitrate (GTN) resulted in concentration-dependent increases in guanylyl cyclase activity provided that the incubations were performed anaerobically and that reduced nicotinamide adenine phosphate was added. Cysteine-dependent activation of guanylyl cyclase by GTN was greater under anaerobic compared to aerobic conditions. Guanylyl cyclase activation by GTN was increased using hepatic microsomes from phenobarbital-treated but not beta-naphthoflavone (BNF)-treated rats and was decreased when microsomes from cimetidine-treated rats were used. The hepatic microsome-dependent activation of guanylyl cyclase by GTN was inhibited by in vitro treatment of microsomes with carbon monoxide, SKF 525A, metyrapone and cimetidine, but not by ranitidine. The sensitivity of isolated rat aorta to the relaxant effects of GTN was increased under low oxygen conditions or when aortae were obtained from phenobarbital- or beta-naphthoflavone-treated rats. Treatment of rats with cimetidine did not affect GTN-induced relaxation. The vascular biotransformation of GTN was increased greater than 3-fold when performed anaerobically, and this increase was prevented by pretreatment of the tissues with carbon monoxide. Together, these data provide strong evidence for the involvement of hepatic cytochromes P-450 in the formation from GTN of an activator of guanylyl cyclase (presumably NO or some closely related compound), and suggest that at least a portion of the vascular biotransformation of GTN is mediated by hemoproteins.


Alpha adrenoceptor subtypes involved in the emetic action in dogs. In order to assess the involvement of alpha-1 and alpha-2 adrenoceptors in emesis, the emetic effect of eight alpha agonists was studied in dogs. The i.m. administration of each agonist elicited dose-dependent emesis. The order of potency in inducing emesis was: clonidine greater than oxymetazoline greater than tramazoline greater than naphazoline greater than xylazine greater than epinephrine greater than methoxamine = phenylephrine. The clonidine-induced emesis was antagonized by adrenoceptor antagonists showing alpha-2 blocking activity, yohimbine, tolazoline and phentolamine. Among these antagonists, yohimbine was the most effective. The alpha-1 and beta adrenergic, cholinergic, dopaminergic, histaminergic, serotonergic and opioid receptor antagonists did not prevent the clonidine-induced emesis. The emesis induced by oxymetazoline, tramazoline, xylazine, naphazoline and epinephrine was also antagonized by a selective alpha-2 adrenoceptor antagonist, yohimbine, but not by a selective alpha-1 adrenoceptor antagonist, prazosin. In contrast, methoxamine and phenylephrine-induced emesis was antagonized by prazosin, but not by yohimbine. Neither yohimbine nor prazosin prevented the morphine- and histamine-induced emesis. These results indicate that alpha-2 adrenoceptors are involved in the mediation of emetic action, and that the alpha adrenoceptor-mediated emesis does not involve beta adrenergic, cholinergic, dopaminergic, histaminergic, serotonergic and opioid receptors in the emetic pathway. This study further suggests that alpha adrenoceptors involved in the emesis are mainly of the alpha-2 type, although the involvement of alpha-1 adrenoceptors cannot be ruled out.


Characterization of the pharmacology of intrathecally administered alpha-2 agonists and antagonists in rats. To examine the pharmacology of the spinal alpha receptor which modulates nociceptive transmission, the antinociceptive effects (52.5 degrees C hot plate; HP) of three i.t. administered alpha-2-preferring agonists [dexmedetomidine (DMET); clonidine (CLON) and ST-91] were determined. The antagonist potency of atipamezole (ATI), idazoxan (IDAZ), yohimbine (YOH) and prazosin (PRA), adrenergic antagonists with differing alpha-2-preferring profiles, were then examined for each of the three agonists. The three agonists produced a dose-dependent block of the HP response with the ED50 and the dose which was just maximally effective being DMET (3.2 and 10 micrograms); CLON (27 and 100 micrograms) and ST-91 (6.1 and 20 micrograms). After determining the time of peak antagonist effect, studies were run in which the just maximally effective dose of each agonist was given in conjunction with one of several doses of the several antagonists. The rank order of potency (and ID50 in microgram) for the several antagonists against each of the three agonists was: DMET = [IDAZ (1.9); ATI (4.1); YOH (70); PRA (greater than 100)]; CLON = [ATI (2.7); IDAZ (23); YOH (52); PRA (greater than 100)]; ST-91 = [PRA (38); YOH (69); ATI (greater than 100); IDAZ (greater than 100)]; where antagonists joined by a common line display overlapping 95% confidence intervals and greater than (greater than) indicates failure to achieve a 50% reversal at the highest antagonist dose.(ABSTRACT TRUNCATED AT 250 WORDS)


Microinjection of the D2 agonist quinpirole into the A10 dopamine region blocks amphetamine-, but not cocaine-stimulated motor activity. Dopamine neurons in the ventral mesencephalon are under the inhibitory influence of dopamine D2 and gamma-aminobutyric acidB receptors. In a previous report, we demonstrated that intra-A10 injections of baclofen, a gamma-aminobutyric acidB agonist, could inhibit the motor-stimulant response to cocaine and amphetamine. In order to further extend these results, we examined the effects of injection of the D2 agonist quinpirole into the A10 region on cocaine- and amphetamine-stimulated motor activity. The results of this study showed that intra-A10 quinpirole dose-dependently decreased locomotor activity. In addition, an intra-A10 injection of 0.3 nmol/microliter quinpirole, a dose chosen for its near threshold effect, could block the motor-stimulant response to a low dose of amphetamine (0.5 mg/kg) and attenuate the response to moderate doses (1.0 and 2.0 mg/kg). Cocaine-stimulated motor activity, at all doses tested (7.5, 15.0 and 30.0 mg/kg), was not altered by intra-A10 quinpirole pretreatment. In vivo microdialysis revealed that quinpirole was unable to block the amphetamine-induced increase in extracellular dopamine concentrations within the nucleus accumbens, despite blocking the motor-stimulant response. It is suggested that the different mechanisms of action of cocaine and amphetamine, uptake blocker vs. releaser or longloop vs. shortloop feedback inhibition of A10 dopamine neurons, respectively, may account for the differential effects that quinpirole had in blocking the motor-stimulant response to these psychostimulants.


Neuronal origins of K(+)-evoked amino acid release from cerebellar cultures. Neuronal cultures from rat cerebellum consisting of approximately 90% glutamatergic granule neurons, 5-7% GABAergic inhibitory interneurons, and 3-5% glial cells, were treated for four days with 50 microM kainic acid (KA) to determine the cellular origin of released endogenous neuroactive substances. KA, known to be selectively toxic to GABAergic neurons, caused an estimated 80% decrease in glutamic acid decarboxylase (GAD) immunofluorescence. Furthermore, K(+)-stimulated release of GABA decreased to 20% of control values, and did not return to control levels in cultures "recovered" two days in KA-free media, suggesting the loss of inhibitory interneurons. Similarly, adenosine and taurine showed decreased K(+)-stimulated release, which was unrecoverable when KA was removed from the medium. K(+)-stimulated release of glutamate and aspartate also decreased by 50% and 70%, respectively, after chronic KA treatment. In contrast, however, this release returned to control levels in recovered cultures. All decreases in K(+)-stimulated release were prevented by concurrent treatment with KA and the KA antagonist 6-cyano-6-nitroquinoxaline-2,3-dione (CNQX), indicating that a receptor-mediated mechanism was involved. We conclude that, in these cultures, most of the K(+)-stimulated release of adenosine and taurine originates from the GABAergic interneurons, the basket and stellate cells, which are selectively killed by the KA treatment. The data also strongly suggest that glutamate and aspartate, the levels of which recover after KA treatment, originate mainly from the granule neurons.