Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.     

Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.     

Distinct interactions between Ca2+/calmodulin and neurotransmitter stimulation of adenylate cyclase in striatum and hippocampus

1. Ca2+ and cAMP both act as intracellular second messengers of receptor activation. In neuronal tissue, Ca2+ acting via calmodulin can elevate cAMP levels. This regulation by Ca2+ provides a means whereby the elevation of intracellular [Ca2+] might modulate cAMP generation. 2. In the present studies, the impact of the Ca2+/calmodulin regulation on receptor-mediated stimulation of activity is compared in striatum and hippocampus--regions of differing sensitivity to Ca2+/camodulin. Ca2+/calmodulin stimulated striatal and hippocampal adenylate cyclase activity by 1.4- and 2.7-fold respectively, while dopamine and vasoactive intestinal peptide (VIP) stimulated the enzyme activity of these respective regions by 1.3- and 2-fold. 3. In the presence of Ca2+/calmodulin, the dopamine dose-response curve in the striatum was shifted upward, without alteration of the slope of the curve or of the maximal stimulation of activity elicited by dopamine. In the hippocampus, the ability of VIP to stimulate adenylate cyclase activity was reduced by the presence of calmodulin. 4. The dose dependence of these actions of calmodulin was examined. In the striatum, the stimulation of adenylate cyclase activity by 0.1 to 0.3 microM calmodulin obscured dopamine stimulation, while 1 to 10 microM was additive with the dopamine stimulation. In the hippocampus, all concentrations of calmodulin (0.1 to 10 microM) reduced VIP-mediated stimulation of enzyme activity. 5. These data suggest that the ratio of calmodulin-sensitive to calmodulin-insensitive adenylate cyclase activity varies in different rat brain regions and that, in those regions in which this ratio is low (e.g., rat striatum and most peripheral systems), calmodulin- and receptor-mediated activation of adenylate cyclase activity will be additive, while in those systems in which this ratio is high (e.g., most of the central nervous system), calmodulin will reduce receptor-mediated stimulation of enzyme activity.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

Cis requirements for alternative splicing of the cardiac troponin T pre-mRNA.

The cardiac troponin T (cTNT) pre-mRNA splices 17 exons contiguously but alternatively splices (includes or excludes) the fifth exon. Because both alternative splice products are processed from the same pre-mRNA species, the cTNT pre-mRNA must contain cis-acting sequences which specify exon 5 as an alternative exon. A cTNT minigene (SM-1) transfected into cultured cells produces mRNAs both including and excluding exon 5. The junctions of exons 4-5-6 and 4-6 in the cTNT minigene mRNAs are identical to those of endogenous cTNT mRNAs and no other exons are alternatively spliced. Thus, the SM-1 pre-mRNA is correctly alternatively spliced in transfected cells. To circumscribe the pre-mRNA regions which are required for the alternative nature of exon 5, we have constructed a systematic series of deletion mutants of SM-1. Transfection of this series demonstrates that a 1200 nt pre-mRNA region containing exons 4, 5, and 6 is sufficient to direct alternative splicing of exon 5. Within this region are two relatively large inverted repeats which potentially sequester the alternative exon via intramolecular base-pairing. Such sequestration of an alternative exon is consistent with models which propose pre-mRNA conformation as being determinative for alternative splicing of some pre-mRNAs. However, deletion mutants which remove the majority of each of the inverted repeats retain the ability to alternatively splice exon 5 demonstrating that neither is required for cTNT alternative splice site selection. Taken together, deletion analysis has limited cis elements required for alternative splicing to three small regions of the pre-mRNA containing exons 4, 5, and 6. In addition, the cTNT minigene pre-mRNA expresses both alternative splice products in a wide variety of cultured non-muscle cells as well as in cultured striated muscle cells, although expression of the cTNT pre-mRNA is normally restricted to striated muscle. This indicates that cis elements involved in defining the cTNT exon 5 as an alternative exon do not require muscle-specific factors in trans to function.     

Use of an androgenized mare as an aid in detection of estrus in mares

A study was conducted over a 2-mo period to compare estrus detection results obtained using an androgenized teaser mare with those obtained with a stallion, using the same group of 10 normally cyclic mares. The teaser mare was androgenized by administration of boldenone undecylenate (500 mg i.m. every 1 to 2 wk), and allowed to run loose with the mare group. Estrus was determined by observation of the group for a 30-min period daily. In the second month of the experiment, a marking harness was used on the androgenized mare to help detect mares mounted when in estrus. Estrous periods detected by each teasing method were 1) first month: stallion, 18; androgenized mare, 5; 2) second month: stallion, 16; androgenized mare, 9. There were no estrous periods detected by the androgenized mare that were not also detected by the stallion. Under these conditions, the androgenized mare was not an adequate estrus detection aid. Also discussed are the successful results of an independent trial on a breeding farm using an androgenized mare as an estrus detection aid.     

The importance of Paracallisoma coecus and myctophid fishes to nesting fork-tailed and Leach's storm-petrels in the Queen Charlotte Islands, British Columbia

The diet of fork-tailed (Oceanodroma furcata) and Leach's storm-petrel(O.leucorhoa) was studied at Petrel Island, a small island northof Hippa Island in the Queen Charlotte Islands, British Columbiain 1983. The diet of the two storm-petrel species consistedmostly of the amphipod Paracallisoma coecus and fish. Most fisheswhich could be identified were myctophids, and may have beenacquired by storm-petrels at night. Fishes increased while P.coecusdeclined in importance as a food over the nestling season ofthe storm-petrels. The amount of oil also declined in the Leach'sstorm-petrel diet over the season. Since P.coecus contains muchoil, the seasonal decline of both P.coecus and oil was probablyassociated. Other less important foods were jellyfishes, copepods,isopods, euphausiids, shrimp, squid and octopus. Jellyfishes(Velella velella) only occurred in the diet of Leach's storm-petrels,which may relate to that storm-petrel feeding in open oceanwhere V. velella occurs most frequently. The most numerous copepodwas Neocalanus cristatus, which was only observed in the storm-petreldiet early on during the study period. An abyssal copepod, Bathycalanusbradyi, and the isopod Cirolana californiensis (tentativelyidentified, the latter only previously known from sea canyonsoff California) were surprising food items of storm-petrels.Most euphausiid prey consisted of Thysanoessa spinifera andEuphausia pacifica.     

Development and analysis of pectic screening media for use in the detection of pectinase mutants

Summary Two solid pectic media were devised for mutually exclusive detection of extracellular polygalacturonase and pectin lyase produced by fungi including the vascular parasite of tomato Verticillium albo-atrum. These media allowed detection of pectinase-defective mutants. Polygalacturonase detection medium contains non-methylated polygalacturonan (sodium polypectate) is buffered at pH 5.0 (Na citrate, 0.05 M) and is calcium-free. In contrast pectin lyase medium contains polymethylgalacturonan (pectin), is buffered at pH 8.0 (HEPES, 0.05 M) and contains calciumrich agar. When glucose was added to the media for selection of catabolite-resistant mutants, enzyme synthesis was still evident, whereas in comparable conditions in liquid culture production was almost completely repressed. This apparent discrepancy is discussed in terms of the influence of basal synthesis, colony biomass and accumulation of oligouronides which repress induced synthesis and activity.Abbreviations CR catabolite repression - CTAB cetyltrimethyl ammoniumbromide - GALA galacturonic acid - NAPP sodium polypectate - PG polygalacturonase - PL pectin lyase - TBA thiobarbituric acid - UGALA unsaturated galacturonic acid     

Loss of striatal mu1 opiate binding by substantia nigra lesions in the rat

Opiate receptors have been identified within the striatum and some have been localized presynaptically to nigrostriatal neurons. Using unilateral ablative lesions of the substantia nigra, we examined binding in the ipsilateral and contralateral striata. Lesions significantly lowered both 3H[D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and 3H[D-Ala2,Leu5]enkephalin (DADL) binding. The inclusion of competitors in these assays revealed a decrease in both mu1 and mu2 receptors. Mu1 binding was slightly more sensitive to the lesioning than mu2 binding. Selective mu1 and mu2 binding assays supported these observations. No change in delta binding was observed in the lesioned striata. These studies raise the possibility that both mu1 and mu2, but not delta, receptors are localized presynaptically on nigrostriatal neurons.