Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.     

Direct repeats flanking the Bacteroides transposon Tn4351 are insertion sequence elements.

The clindamycin-erythromycin resistance (Ccr Emr) region of the Bacteroides transposon Tn4351 is flanked by direct repeats. This study showed that the direct repeats are insertion sequence (IS) elements. Although both IS elements can mediate transfer of the chloramphenicol (Cmr) marker on pBR328 by cointegrate formation with the conjugal IncW plasmid R388, IS4351R-mediated transfer of Cmr occurred at a consistently lower frequency than did the transfer mediated by IS4351L. Analysis of plasmids from the resultant transconjugants revealed IS-mediated activities such as deletions, tandem duplication of IS4351L, and excision of IS4351R.     

Carbon Balance of a Mannitol Fermentation and the Biosynthetic Pathway

The carbon balance was determined for a fermentation in which mannitol is produced from glucose by an Aspergillus species. The products found were: cells (17% of carbon input), CO(2) (26%), mannitol (35%), glycerol (10%), erythritol (2.5%), glycogen (1%), and unidentified compounds (8%). Thus, 92% of the carbon input was accounted for. Cell-free enzyme studies showed that mannitol was synthesized via the reduction of fructose-6-phosphate and not by the direct reduction of fructose. If the cell yield from glucose was assumed to be 50% and the theoretical conversion efficiency from glucose to polyols was 90%, as calculated from the energy balance, then 34% of the glucose carbon was used for growth and 53% was used for polyol formation.     

Analysis of peptides from known proteins: Clusterization in sequence space

A combinatorial sequence space (CSS) model was introduced to represent sequences as a set of overlapping k-tuples of some fixed length which correspond to points in the CSS. The aim was to analyze clusterization of protein sequences in the CSS and to test various hypotheses about the possible evolutionary basis of this clusterization. The authors developed an easy-to-use technique which can reveal and analyze such a clusterization in a multidimensional CSS. Application of the technique led to an unexpectedly high clusterization of points in the CSS corresponding to k-tuples from known proteins. The clusterization could not be inferred from nonuniform amino acid frequencies or be explained by the influence of homologous data. None of the tested possible evolutionary and structural factors could explain the clusterization observed either. It looked as if certain protein sequence variations occurred and were fixed in the early course of evolution. Subsequent evolution (predominantly neutral) allowed only a limited number of changes and permitted new variants which led to preservation of certain k-tuples during the course of evolution. This was consistent with the theory of exon shuffling and protein block structure evolution. Possible applications of sequence space features found were also discussed.Correspondence to: H.A. Lim     

Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates. Studies of manganese complexes by NMR relaxation rates of water protons.

The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The binding of Mn(II) to the enzyme was investigated by EPR. The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8. The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP. PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not at all to (E-P)-Mn.Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement. No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement. However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA. An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively. The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon. Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.     

Uptake of the components of phenylalanylphenylalanine and maltose by intestinal epithelium

The observed rate of phenylalanine absorption into rat intestinal rings with 0.5 or 5.0 mM phenylalanine is greater than that for absorption of phenylalanine from 0.25 or 2.5 mM Phe-Phe, respectively. With the amino acid phenylalanine, V for absorption is the same whether Na⁺ is present (149 mM) or absent, but the concentration at which the half-maximal transport rate occurred (K_t) is greater in the absence of Na⁺. For Phe-Phe, the V decreases in the absence of Na⁺ whilst K_t is not influenced by the Na⁺ concentration. The different effect of Na⁺ on Phe and Phe-Phe transport indicates that the absorptive mechanism for Phe-Phe is different from that for phenylalanine. Absorption of a mixture of [U-¹⁴C]Phe-Phe and Phe-[G-³H]Phe showed identical rates of uptake of the carboxyl and amino terminal amino acids.Studies of transport of radioactive maltose showed that the rates of uptake of the reducing and non-reducing glucosyl moieties are identical. Radioactive maltose absorption is not inhibited by glucose oxidase.These results provide evidence that in intestinal epithelium, hydrolysis of Phe-Phe and maltose does not occur on the cell surface with release of the hydrolyzed products to the medium. Rather, hydrolysis and release of the reaction products occur at a point on the cytosol side of a diffusion barrier located in the brush border membrane.     

Isolation and characterization of two novel rat ovarian lactogen receptor cDNA species

Two novel lactogen receptor cDNA clones (2.1 and 1.2 kb) were isolated from a rat ovarian cDNA library. Nucleotide sequence of the 2.1 kb clone codes for a 610 aa receptor (nonglycosylated mol. wgt. 66,000 D) with an extracellular domain, a transmembrane region and an intracellular domain, and exhibited significant overall similarity with the rat liver receptor (310 aa) and both rabbit mammary and human hepatoma receptors (616 and 622 aa). However, the ovarian lactogen receptor sequence contains a unique cytoplasmic domain of 110 aa and consensus sequences for both a tyrosine phosphorylation site and an ATP/GTP type A binding site, and thus has potential for signal transduction and mitogenic activity. The 1.2 kb clone codes for a truncated binding form of 150 aa that is identical with the ovarian long form over only the first 130 residues, and lacks the transmembrane region. Differences between long and short forms of the ovarian lactogen receptors and the truncated liver species may result from alternative splicing. The prolactin holoreceptor gene(s) has the potential for producing several receptor subtypes that differ in tissue-specific expression, size, compartmentalization and mode of signal transduction, and may subserve the divergent functions of prolactin in its several target cells.