β-Sheet Models for the Ordered Filamentous Structure Formed by a Peptide That Enhances the Action of Insulin

Certain peptides with sequences related to part of the major histocompatibility complex class I antigen enhance the action of insulin. These peptides also aggregate into fibrous structures that seem to be related to their biological activity. In the current study, the 17-residue peptide with amino acid sequence Gly-Asn-Glu-Gln-Ser-Phe-Arg-Val-Asp-Leu-Arg-Thr-Leu-Leu-Arg-Tyr-Ala is used as a representative example of these bioactive molecules. As seen by electron microscopy, the peptide associates into gently twisted ribbons, 50 Å thick, in which the amount of twist decreases as the ribbons become wider. X-ray diffraction analysis suggests that the peptides are arranged as in an antiparallel β-sheet extending essentially endlessly along the fiber axis. The amino acid sequence of the peptide is such that one side of the β-sheet is predominantly polar while the opposite side is nonpolar. This allows the β-sheets to form multilayers with alternating hydrophobic and hydrophilic interfaces. The length of the extended peptide (≈54 Å) determines the thickness of the ribbon and the tendency of individual β-sheets to twist accounts for the twisting of the ribbons. An alternative model is also discussed, again based on antiparallel β-sheets, but with adjacent sheets interdigitated in a “side-by-side” fashion rather than forming stacked layers. Comparable inactive peptides such as Gly-Asn-Glu-Gln-Ser- A_l_a_-Arg-Val-Asp-Leu-Arg-Thr-Leu-Leu-Arg-Tyr-T_y_r_ (changed amino acids underlined) do not form ordered filamentous structures.     

Activity of single-stranded DNA endonucleases in mung bean is associated with cell division

A single-strand-specific endonuclease from mung bean sprouts is widely usedin molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.     

Characterization of a novel plant poly(A) polymerase

We have purified and characterized poly(A) polymerases (PAPs) from Pisum sativum, Brassica juncea, and Zea mays. Through chromatography on DEAE-Sepharose and heparin-Sepharose, these PAPs copurified as a single enzyme along with RNPs that could provide RNA substrates for the enzyme. More extensive purification by chromatography on MonoQ resulted in the resolution of the PAPs into as many as three fractions. One of these (PAP-I) contained a 43-kDa polypeptide immunologically related to the yeast PAP, and two others (PAP-II and PAP-III) contained RNAs that could serve as substrates for polyadenylation. These fractions by themselves possessed little PAP activity, but mixtures containing combinations of these displayed substantial activity. Similar PAP factors (PAP-I and PAP-III) were identified after fractionation of extracts prepared from Brassica juncea and Zea mays. The factors from one plant were completely interchangeable with those from different plants. We conclude that the poly(A) polymerases present in vegetative plant tissues consist of more than one component. In this respect, they are substantially different from other reported plant, mammalian, and yeast PAPs.     

How Does the Crayfish Swimmeret System Work? Insights from Nearest-Neighbor Coupled Oscillator Models

Rhythmic movements of crayfish swimmerets are coordinated by a neural circuit that links their four abdominal ganglia. Each swimmeret is driven by its own small local circuit, or pattern-generating module. We modeled this networkas a chain of four oscillators, bidirectionally coupled to their nearest neighbors, and tested the models ability to reproduce experimentally observed changes in intersegmental phases and in period caused by differential excitation of selected abdominal ganglia. The choices needed to match the experimental data lead to the followingpredictions: coupling between ganglia is asymmetric; the ascending and descending coupling have approximately equal strengths; intersegmental coupling does not significantly affect the frequency of the system; and excitation affects the intrinsic frequencies of the oscillators and might also change properties ofintersegmental coupling.     

Measurement of 15N-1H coupling constants in uniformly 15N-labeled proteins: Application to the photoactive yellow protein

A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 117³J(NH) coupling constants. In addition, 29³J(NH^(i–1)) coupling constantscould be measured, providing information about the backbone angle .These data, in conjunction with the magnitudes of the³J(H^NH) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theH resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate H resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the H resonances for these residues. In addition,reliable and precise , dihedral constraints were also derived forthese residues from the J-coupling data.     

The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.     

Intratracheal instillation of a novel NO/nucleophile adduct selectively reduces pulmonary hypertension

Brilli, Richard J., Brian Krafte-Jacobs, Daniel J. Smith,Dominick Roselle, Daniel Passerini, Amos Vromen, Lori Moore, CsabaSzabó, and Andrew L. Salzman. Intratracheal instillation ofa novel NO/nucleophile adduct selectively reduces pulmonary hypertension. J. Appl. Physiol. 83(6):1968-1975, 1997.We examined the pulmonary and systemichemodynamic effects of administering soluble nitric oxide (NO) donorcompounds (NO/nucleophile adducts, i.e., NONOates) directly into thetrachea of animals with experimentally induced pulmonary hypertension.Steady-state pulmonary hypertension was created by using thethromboxane agonist U-46619. Yorkshire pigs were randomly assigned toone of four groups: group 1,intratracheal saline (control; n = 8);group 2, intratracheal sodiumnitroprusside (n = 6);group 3, intratracheal ethylputreanineNONOate (n = 6); andgroup 4, intratracheal2-(dimethylamino)-ethylputreanine NONOate (DMAEP/NO;n = 6). Pulmonary and systemichemodynamics were monitored after drug instillation.Group 4 had significant reductions in pulmonary vascular resistance index (PVRI) at all time points comparedwith steady state and compared with group1 (P < 0.05), whereas systemic vascular resistance index did not change. The meanchange in mean pulmonary arterial pressure in group4 was 33.1 ± 1.2% compared with +6.4 ± 1.3% in group 1 (P < 0.001), and the mean change inmean arterial pressure was 9.3 ± 0.7% compared with acontrol value of 0.9 ± 0.5%(P < 0.05). Groups 2 and 3 hadsignificant decreases in both PVRI and systemic vascular resistanceindex compared with steady state and with group1. In conclusion, intratracheal instillation of apolar-charged tertiary amine NONOate DMAEP/NO results in the selectivereduction of PVRI. Intermittent intratracheal instillation of selectiveNONOates may be an alternative to continuously inhaled NO in thetreatment of pulmonary hypertension.

     

Crystallization and X-ray analysis of a single fab binding domain from protein L of Peptostreptococcus magnus

Protein L is a multi domain cell wall constituent of certain strains of Peptostreptococcus magnus which binds to the variable domain of immunoglobulin κ-light chains. A single immunoglobulin-binding domain of M_r = 9000 from this protein has been isolated and crystallized. The crystals are of space group P4₂2₁2, with cell dimensions a = b = 66.9 Å, c = 68.3 Å, and diffract to at least 2.2 Å resolution. The asymmetric unit of the crystal contains two molecules of the protein L domain, related by a noncrystallographic 2-fold axis, as revealed by a self-rotation function calculated with native diffraction data. © 1995 Wiley-Liss, Inc.