Conductance Ratios and Cellular Identity

Recent experimental evidence suggests that coordinated expression of ion channels plays a role in constraining neuronal electrical activity. In particular, each neuronal cell type of the crustacean stomatogastric ganglion exhibits a unique set of positive linear correlations between ionic membrane conductances. These data suggest a causal relationship between expressed conductance correlations and features of cellular identity, namely electrical activity type. To test this idea, we used an existing database of conductance-based model neurons. We partitioned this database based on various measures of intrinsic activity, to approximate distinctions between biological cell types. We then tested individual conductance pairs for linear dependence to identify correlations. Contrary to experimental evidence, in which all conductance correlations are positive, 32% of correlations seen in this database were negative relationships. In addition, 80% of correlations seen here involved at least one calcium conductance, which have been difficult to measure experimentally. Similar to experimental results, each activity type investigated had a unique combination of correlated conductances. Finally, we found that populations of models that conform to a specific conductance correlation have a higher likelihood of exhibiting a particular feature of electrical activity. We conclude that regulating conductance ratios can support proper electrical activity of a wide range of cell types, particularly when the identity of the cell is well-defined by one or two features of its activity. Furthermore, we predict that previously unseen negative correlations and correlations involving calcium conductances are biologically plausible.     

Detection of femtomolar quantities of the ganglioside GM1 on thin-layer chromatography plates by native choleratoxin and labeled antisera

After separation of gangliosides by thin-layer chromatography, femtomolar quantities of G_M1 were detected by incubating the plate with native choleratoxin, followed by anticholeratoxin and species-specific labeled antiserum. Only stable reagents were involved when antiserum labeled with horseradish peroxidase was used. Native choleratoxin rather than iodinelabeled toxin ensured good reproducibility of the method.     

Ribonuclease P: the diversity of a ubiquitous RNA processing enzyme

Ribonuclease P is the endonuclease required for generating the mature tRNA 5'-end. The ribonucleoprotein character of this enzyme has now been proven in most organisms and organelles. Exceptions, however, are still the chloroplasts, plant nuclei and animal mitochondria where no associated RNAs have been detected to date. In contrast to the known RNA subunits, which are fairly well-conserved in size and structure among diverse phylogenetic groups, the protein contribution to the holoenzyme is highly variable in size and number of the individual components. The structure of the bacterial protein component has recently been solved. In contrast, the spatial arrangement of the multiple subunits in eukaryotic enzymes is still enigmatic. Substrate requirements of the enzymes or their catalytic RNA subunits are equally diverse, ranging from simple single domain mimics to an almost intact three-dimensional structure of the pre-tRNA substrate. As an example for an intermediate in the enzyme evolution, ribonuclease P from the Cyanophora paradoxa cyanelle will be discussed in more detail. This enzyme is unique, as it combines cyanobacterial and eukaryotic features in its function, subunit composition and holoenzyme topology.     

cDNA sequence and tissue distribution of the mRNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (calpactin I)

We have isolated and sequenced cDNA clones of bovine and murine p11 mRNAs. The nonpolyadenylated mRNAs are predicted to be 614 and 600 nucleotides, respectively. The p11 mRNAs both contain a 291 nucleotide open reading frame, preceded by a 5'-untranslated region of 73 nucleotides in bovine p11 mRNA and of 68 nucleotides in murine p11 mRNA. The deduced bovine p11 amino acid sequence is identical to the previously published partial bovine and complete porcine p11 protein sequence except for an additional COOH-terminal lysine residue. The bovine and murine p11 proteins are 92% homologous, whereas at the nucleotide level the conservation is 89% in the coding region and 75% in the 3'-untranslated region. Southern analysis of murine genomic DNA detected a single p11 gene, less than 10 kilobase pairs in size, containing as many as three introns. The p11 gene has been assigned to mouse chromosome 3 by analysis of interspecific hybrid cell panels and recombinant inbred mouse strains. The p11 gene is closely linked to the Xmmv-65 endogenous leukemia virus env gene and the guanylate binding protein-1 gene. Northern analyses of RNAs from mouse tissues and cell lines indicated that p11 mRNA levels vary widely. They are very low in liver, heart, and testes, moderate in brain, spleen, and thymus, and high in kidney, intestine, and lung. Analysis of the same RNA samples for p36 mRNA levels showed that expression of p11 and p36 mRNAs is not always coordinated. Brain and the mouse embryonal carcinoma cell line F9 contain moderate to high levels of p11 mRNA with very low levels of p36 mRNA. Sequence homology between p11 and the S100 proteins, and the serum-induced 2A9 gene product, as well as possible functions of p11 are discussed.     

cDNA structure of murine C4b-binding protein, a regulatory component of the serum complement system

A cDNA library representing total poly(A+) RNA from the livers of male B10.WR mice was screened with a 1097 base pair (bp) probe obtained from a partial human C4b-binding protein (C4BP) cDNA clone. Two cDNA clones were isolated, the largest of which was sequenced and found to be 1889 bp in length exclusive of the poly(A) tail. The predicted mouse C4BP polypeptide chain encoded by 1239 bp is 413 amino acid residues in length and has a calculated molecular weight of 45,281. The 370-nucleotide sequence upstream from the codon for the predicted amino terminus contains two possible in-phase translational start signals which yield leader sequences of 56 and 13 amino acid residues, respectively. The 3'-untranslated region is 277 bp long, and there are two potential overlapping poly(A) recognition signals, AATTAA and ATTAAAA, located 26 and 25 bp, respectively, upstream from the poly(A) tail; these are preceded by five other potential polyadenylation signals. Beginning at the amino terminus and continuing through to residue 358, there are six contiguous regions of internal homology, each about 60 amino acids in length. The carboxy-terminal 55 amino acid sequence shares no homology with the repeating units. Extensive homology was found with human C4BP at the amino acid level (61%) as well as at the nucleotide level for both the coding and 3'-untranslated regions. Significant differences, however, were observed between mouse and human C4BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple and rapid preparation of M13 sequencing templates for manual and automated dideoxy sequencing.

A simple and rapid procedure for the preparation of M13 single stranded DNA sequencing templates which does not involve phenol extractions and alcohol precipitations is described. Bacteriophages are precipitated from media supernatants with acetic acid and recovered on glass fiber filters. Subsequent dissociation of the phages and removal of contaminants is performed while the DNA is bound to the glass. Finally, the purified DNA is eluted in a small volume of low-salt buffer. The yield is higher than that obtained by standard methods. The simplified procedure takes less than 30 minutes and does not demand special skills or equipment; the sequence resolution is as good as that obtained by standard procedures both with the Klenow fragment and T7 DNA polymerase, with radioactive labelling as well as in automated sequencing with a fluorescent label.     

Glucose and amino acid metabolism in chick telencephalon slices: Changes with incubation conditions and animals' development

Glucose and amino acid metabolism in 1- and 30-day-old chick telencephalon slices was studied in two incubation media in the presence or in the absence of a continuous oxygenation. Medium 1 has a composition and a tonicity similar to cerebrospinal fluid, medium 2 is hypertonic and does not contain any K⁺ ions. The incorporation of glucose carbon into amino acids and the distribution of radioactivity between the different amino acids are close to the ones observed in the chick brain in vivo only when the slices are incubated in medium 1, with oxygen at 30 days and without oxygen for the 1-day-old chick. It also appears that if oxygenation is necessary for incubation of mature brain tissue in vitro, the absence of the medium oxygenation is more suitable for the study of glucose metabolism in 1-day-old chick brain slices.     

Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat

Summary The observation that tissue-type plasminogen activator (tPA) activity increased dramatically in preovulatory follicles has led to the hypothesis that plasminogen activation is causally related to follicle rupture. With immunohistochemistry, we have studied the appearance of tPA in ovaries of immature rats induced to ovulate and in adult cycling rats. Treatment of immature female rats with a single dose of pregnant mare serum gonadotropin (PMSG) induced follicular maturation. A subsequent human chorionic gonadotropin (hCG) injection resulted in follicle rupture 12–14 h later. PMSG treatment alone did not induce appearance of tPA-immunoreactive cells in any ovarian compartment. After hCG stimulation, however, theca cells, granulosa cells, and oocytes of pre- and postovulatory follicles displayed distinct tPA immunoreactivity. Fibroblastlike cells in the theca layers and tunica albuginea of the follicle apex also demonstrated localized cytoplasmic tPA reactivity. In addition to tPA synthesis in preovulatory follicles, hCG also induced tPA staining in the theca (but not granulosa) layers of non-ovulatory follicles. At 24 h after hCG treatment, there was a marked tPA staining in developing corpora lutea, ovulated ova, and oviductal epithelium. Ovaries from regularly cycling adult rats displayed a similar ovulation-related pattern of tPA immunostaining. The appearance of tPA in different cell types of the preovulatory follicle and in the fibroblast-like cells at the follicle apex, strengthens the hypothesis of a direct involvement of tPA in follicle rupture. Presence of tPA in postovulatory oocytes, cumulus cells, and surrounding oviductal epithelium may also indicate a role for tPA in the transfer of eggs in the oviduct.This work was supported by NIH Research Grants HD-14084; 12303