NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit-specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area-specific rearrangement of NR2B subunit expression following birth.     

Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs

Theodorou, Andreas, Natalie Weger, Kathleen Kunke, KyooRhee, David Bice, Bruce Muggenberg, and Richard Lemen. Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs. J. Appl. Physiol.83(3): 912-917, 1997.In ragweed (RW)-sensitized beagle dogs, wetested the hypothesis that reactivity of the pulmonary vasculature wasenhanced with aerosolized histamine (Hist) and RW. Seven dogs wereneonatally sensitized with repeated intraperitoneal RW injections, and12 dogs were controls (Con). The dogs were anesthetizedwith intravenous chloralose, mechanically ventilated, and instrumentedwith femoral arterial and pulmonary artery catheters. Specific lungcompliance(CL_sp),specific lung conductance (G_sp),systemic vascular resistance index, and pulmonary vascular resistanceindex (PVRI) were measured before and after bronchoprovocation withHist and RW. After Hist inhalation (5 breaths of 30 mg/ml), both Conand RW dogs had significant (P < 0.05) decreases inCL_sp(51 ± 4 and 53 ± 5%, respectively) andG_sp (65 ± 5 and69 ± 3%, respectively), but only RW-sensitized dogs had asignificant increase in PVRI (38 ± 10%). After RW inhalation (60 breaths of 0.8 mg/ml), only RW-sensitized dogs had significant increases (62 ± 20%) in PVRI and decreases inG_sp (77 ± 4%) and CL_sp(65 ± 7%). We conclude that, compared with Con,RW-sensitized beagle dogs have increased pulmonary vasoconstrictiveresponses with Hist or RW inhalation.

     

Solvent polarity-dependent structural refolding: A CD and NMR study of a 15 residue peptide

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a β-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular β-sheet to a helical secondary structure is known as “switch” behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the “switch.” The tetrad has a strong β-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3₁₀-helix suggesting that the tetrad adopts a type III β-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3₁₀-helix at a critical polarity favoring intrachain hydrogen bonds. © 1995 Wiley-Liss, Inc.     

Developmental regulation of presence of binding sites for neoglycoproteins and endogenous lectins in various embryonic stages of human lung,liver and heart

Protein-carbohydrate interactions are supposed to play key roles in the mechanisms of cell adhesion, biosignalling and intracellular routing, warranting the analysis of the developmental course of expression of epitopes of this system. Thus, a panel of carrier-immobilized carbohydrate ligands was used as probes, namely lactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, fucose and maltose. Additionally, an antibody to an endogenous -galactoside-binding lectin (anti-galectin-1), the biotinylated lectin and two further human lectins, namely the macrophage migration inhibitory factor-binding sarcolectin and serum amyloid P component (SAP) that displays selectivity for sulphated sugars and mannose-6-phosphate, were included. They enabled us to assess the extent of the presence of respective binding sites in fixed sections from human lungs (pulmonary epithelial cells), livers (hepatocytes) and hearts (myocard cells) of 10–50 weeks gestation. Invariably, specific binding was detected in the three organ types, at least in certain stages. In most of the cases, the intensity of staining exhibited developmental regulation. The apparent patterns reveal similarities between the different cell types, as seen with immobilizedN-acetylglucosamine as well as with labelled galectin-1 and sarcolectin. However, drastic differences among such patterns with nearly opposite developmental courses do also occur, as detected for carrier-attached mannose and maltose residues. These results point to a potential importance for the detected glycohistochemical features in human development and substantiate the possibility of differential regulation of the presence of binding sites for distinct sugars within a certain organ and between the individual cell types of the monitored organs.     

Decavanadate is responsible for vanadate-induced two-dimensional crystals in sarcoplasmic reticulum

Two-dimensional protein crystals of the calcium pump protein of sarcoplasmic reticulum (SR) from fast skeletal muscle were induced using Na₃VO₃ as first described by Dux and Martonosi. These crystals exhibit repeat rows 11 nm apart which contain discrete units with 7 nm repeats. Four different methods of sample preparation for electron microscopy, i.e., negative staining, freezedrying, freeze-fracturing, and thin-sectioning electron microscopy, each give complimentary repeat units. The SR-membrane crystals exhibit surface structure by the freeze-drying technique and row-like structures on the normally smooth outer face of normal SR. The formation of the membrane crystals is dependent on the pH and concentration of the vanadate. Only conditions favoring the presence of decavanadate yield crystals. At low concentrations and neutral pH, decavanadate is unstable and with time converts to smaller oligomers and the monomer. The presence of membrane crystals was correlated with the life span of the decavanadate. Membrane crystals were obtained in the SR membrane from fast twitch muscle from light and heavy SR, referable to longitudinal and terminal cisternae as well as from reconstituted SR. Canine cardiac SR did not crystallize under these conditions.Abbreviations Tris (tris[hydroxymethyl])aminomethane - TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), 2-(2-hydroxy-1-bis[hydroxymethyl]ethyl)aminoethanesulfonic acid - SR sarcoplasmic reticulum - CPP calcium pump protein Dedicated to the memory of Prof. David E. Green, friend, mentor, and colleague.     

Can a Ca2+ pump in the endoplasmic reticulum of the Lepidium root be the trigger for rapid changes in membrane potential after gravistimulation?

Since gravistimulation is followed by alterations in the external current symmetry (Behrens et al., 1982), the effect of gravistimulation on cellular membrane potential was investigated using conventional glass microelectrode techniques. The resting potential of statocytes in a vertically oriented root is approx. -118 mV. Upon gravistimulation, the membrane potential is temporarily depolarized (lag time = 2 s) to a potential of approx. -93 mV. This depolarization is only observed in statocytes located on the physically lower root flank while those on the corresponding upper flank become weakly hyperpolarized (approx. -13 mV). These results reflect altered ion fluxes across the plasma membrane. The perception of gravistimulus was suggested to result from a pressure of the amyloplasts on the distal endoplasmic reticulum (ER) of the statocytes (Sievers and Volkmann, 1972). A causal relationship between changes in ER-amyloplast interactions and the rapid alterations in plasma membrane potential described above is not known. A candidate for such an intracellular messenger is Ca2+. As a first step in establishing the validity of such an assumption, we have isolated ER membranes from roots. When incubated with micromolar concentrations of Ca2+, the vesicular membrane fraction accumulates Ca2+. The accumulation is ATP-dependent and -specific and is directly coupled to ATP hydrolysis since a protonophore shows no inhibitory effect. Thus, in analogy to the sarcoplasmic reticulum of muscle, regulation of an ER-localized Ca2+ compartment might be an important step in such complex processes as stimulus-transduction in gravitropism.     

Effects of prolonged omnilateral gravistimulation on the ultrastructure of statocytes and on the graviresponse of roots

Statocytes of vertically growing roots of Lepidium sativum L. exhibit a strict polarity: The nucleus is positioned near the proximal periclinal cell wall, amyloplasts are sedimented on a complex of rough endoplasmic reticulum (ER) consisting of parallel cisternae near the distal periclinal cell wall.When 24 h old, vertically grown roots are rotated for an additional 20 h on a horizontal clinostat, this polarity is destroyed. Furthermore, the prolonged omnilateral stimulation leads to a damage of the statocytes, which in some cases ends in the self-destruction of the sensitive cells. The different components of the ultrastructural respones of the statocytes are: Displacement of the nucleus; changes in amount and distribution of the ER; loss of amyloplast starch; confluence of lipid droplets to large aggregates: a considerable increase of the lytic compartment. In addition, even anticlinal cell walls may be lysed up to small stumps. As all these effects are clearly restricted to the statocytes, only these cells are able to respond to the continuously changing direction of the gravity vector, thus perceiving gravity as such.After being exposed horizontally, the graviresponse of rotated roots is delayed as compared to the controls. About 20% of the rotated roots do not respond (curve) at all, but grow perpendicular in relation to the gravity vector. Perception of gravity is inevitably correlated with the polarity and the integrity of the statocytes.Abbreviation ER endoplasmic reticulum A preliminary report was presented at the Fall Meeting of the German Society for Cell Biology in Salzburg, Austria, September 1979 (Hensel and Sievers 1979)This paper represents part of a dissertation (D 5) of W. H.