Dynamic reconstruction of eye closure by muscle transposition or functional muscle transplantation in facial palsy

For patients with facial palsy, lagophthalmus is often a more serious problem than the inability to smile. Dynamic reconstruction of eye closure by muscle transposition or by free functional muscle transplantation offers a good solution for regaining near-normal eye protection without the need for implants. This is the first quantitative study of three-dimensional preoperative and postoperative lid movements in patients treated for facial paralysis. Between February of 1998 and April of 2002, 44 patients were treated for facial palsy, including reconstruction of eye closure. Temporalis muscle transposition to the eye was used in 34 cases, and a regionally differentiated part of a free gracilis muscle transplant after double cross-face nerve grafting was used in 10 cases. Patients' facial movements were documented by a three-dimensional video analysis system preoperatively and 6, 12, 18, and 24 months postoperatively. For this comparative study, only the data of patients with preoperative and 12-month postoperative measurements were included. In the 27 patients with a final result after temporalis muscle transposition for eye closure, the distance between the upper and lower eyelid points during eye closing (as for sleep) was reduced from 10.33 +/- 2.43 mm (mean +/- SD) preoperatively to 5.84 +/- 4.34 mm postoperatively on the paralyzed side, compared with 0.0 +/- 0.0 mm preoperatively and postoperatively on the contralateral healthy side. In the resting position, preoperative values for the paralyzed side changed from 15.11 +/- 1.92 mm preoperatively to 13.46 +/- 1.94 mm postoperatively, compared with 12.17 +/- 2.02 mm preoperatively and 12.05 +/- 1.95 mm postoperatively on the healthy side. In the nine patients with a final result after surgery using a part of the free gracilis muscle transplant reinnervated by a zygomatic branch of the contralateral healthy side through a cross-face nerve graft, eyelid closure changed from 10.21 +/- 2.72 mm to 1.68 +/- 1.35 mm, compared with 13.70 +/- 1.56 mm to 6.63 +/- 1.51 mm preoperatively. The average closure for the healthy side was from 11.20 +/- 3.11 mm to 0.0 +/- 0.0 mm preoperatively and from 12.70 +/- 1.95 mm to 0.0 +/- 0.0 mm postoperatively. In three cases, the resting tonus of the part of the gracilis muscle transplant around the eye had increased to an extent that muscle weakening became necessary. Temporalis muscle transposition and free functional muscle transplantation for reanimation of the eye and mouth at the same time are reliable methods for reconstructing eye closure, with clinically adequate results. Detailed analysis of the resulting facial movements led to an important improvement of the authors' operative techniques within the last few years. Thus, the number of secondary operative corrections could be significantly reduced. These qualitative and quantitative studies of the reconstructed lid movements by three-dimensional video analysis support the authors' clinical concept of temporalis muscle transposition being the first-choice method in adult patients with facial palsy. In children, free muscle transplantation is preferred for eye closure, so as not to interfere with the growth of the face by transposition of a masticatory muscle. In addition, a higher degree of central plasticity in children might be expected.     

13C solid-state NMR chemical shift anisotropy analysis of the anomeric carbon in carbohydrates

(13)C NMR solid-state structural analysis of the anomeric center in carbohydrates was performed on six monosaccharides: glucose (Glc), mannose (Man), galactose (Gal), galactosamine hydrochloride (GalN), glucosamine hydrochloride (GlcN), and N-acetyl-glucosamine (GlcNAc). In the 1D (13)C cross-polarization/magic-angle spinning (CP/MAS) spectrum, the anomeric center C-1 of these carbohydrates revealed two well resolved resonances shifted by 3-5ppm, which were readily assigned to the anomeric alpha and beta forms. From this experiment, we also extracted the (13)C chemical shift anisotropy (CSA) tensor elements of the two forms from their spinning sideband intensities, respectively. It was found out that the chemical shift tensor for the alpha anomer was more axially symmetrical than that of the beta form. A strong linear correlation was obtained when the ratio of the axial asymmetry of the (13)C chemical shift tensors of the two anomeric forms was plotted in a semilogarithmic plot against the relative population of the two anomers. Finally, we applied REDOR spectroscopy to discern whether or not there were any differences in the sugar ring conformation between the anomers. Identical two-bond distances of 2.57A (2.48A) were deduced for both the alpha and beta forms in GlcNAc (GlcN), suggesting that the two anomers have essentially identical sugar ring scaffolds in these sugars. In light of these REDOR distance measurements and the strong correlation observed between the ratio of the axial asymmetry parameters of the (13)C chemical shift tensors and the relative population between the two anomeric forms, we concluded that the anomeric effect arises principally from interaction of the electron charge clouds between the C-1-O-5 and the C-1-O-1 bonds in these monosaccharides.     

Vaccinia virus 4c (A26L) protein on intracellular mature virus binds to the extracellular cellular matrix laminin

Vaccinia virus intracellular mature virus (IMV) binds to glycosaminoglycans (GAGs) on cells via three virion proteins, H3L, A27L, and D8L. In this study, we demonstrated that binding of IMV to BSC40 cells was competitively inhibited by soluble laminin but not by fibronectin or collagen V, suggesting that this cell surface extracellular matrix (ECM) protein may play a role in vaccinia virus entry. Moreover, IMV infection of GAG(-) sog9 cells was also inhibited by laminin, demonstrating that virion binding to laminin does not involve a prior interaction with GAGs. Furthermore, comparative envelope protein analyses of wild-type vaccinia virus strain Western Reserve, which binds to laminin, and of a mutant virus, IA27L, which does not, showed that the A26L open reading frame (ORF), encoding an envelope protein, was mutated in IA27L, resulting in A26L being absent from the IMV. Expression of the wild-type A26L ORF in IA27L resulted in laminin binding activity. Moreover, recombinant A26L protein bound to laminin in vitro with a high affinity, providing direct evidence that A26L is the laminin binding protein on IMV. In summary, these results reveal a novel role for the vaccinia viral envelope protein A26L in binding to the ECM protein laminin, an association that is proposed to facilitate IMV entry.     

Vaccinia Viral Protein A27 Is Anchored to the Viral Membrane via a Cooperative Interaction with Viral Membrane Protein A17

The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18–50 residues) and C-terminal (162–203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (K_A = 3.40 × 10⁸ m⁻¹) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (K_A = 3.40 × 10⁵ m⁻¹), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely ³²SFMPK³⁶ (denoted as F1 binding) and ²⁰LDKDLFTEEQ²⁹ (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.     

1H, 15N, and 13C resonance assignments for a monomeric mutant of the HIV-1 capsid protein

The mature fullerene cone-shaped capsid of the human immunodeficiency virus 1 is composed of about 1,500 copies of the capsid protein (CA). The CA is 231 residues long, and consists of two distinct structural domains, the N-terminal domain and the C-terminal domain (CTD), joined by a flexible linker. The wild type CA exhibits monomer-dimer equilibrium in solution through the CTD-CTD dimerization. This CTD-CTD interaction, together with other intermolecular interdomain interactions, plays significant roles during the assembly of the mature capsid. In addition, CA-CA interactions also play a role in the assembly of the immature virion. The CA also interacts with some host cell proteins within the viral replication cycle. Thus, the capsid protein has been of significant interest as a target for designing inhibitors of assembly of immature virions and mature capsids and inhibitors of its interactions with host cell proteins. However, the equilibrium exhibited by the wild-type CA protein between the monomeric and dimeric states, along with the inherent flexibility from the interdomain linker, have hindered attempts at structural determination by solution NMR and X-ray crystallography methods. In this study, we have utilized a CA protein with W184A and M185A mutations that abolish the dimerization of CA protein as well as its infectivity, but preserve most of the remaining properties of the wild type CA. We have determined the detailed solution structure of the monomeric W184A/M185A-CA protein using 3D-NMR spectroscopy. Here, we present the detailed sequence-specific NMR assignments for this protein.     

Multiple conformations of the metal-bound pyoverdine PvdI, a siderophore of Pseudomonas aeruginosa: a nuclear magnetic resonance study

Under iron-deficient conditions, the Gram-negative bacterium Pseudomonas aeruginosa ATCC 15692 secretes a peptidic siderophore, pyoverdine PvdI, composed of an aromatic chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and a partially cyclized octapeptide, d-Ser- l-Arg- d-Ser- l-FoOHOrn-( l-Lys- l-FoOHOrn- l-Thr- l-Thr), in which the C-terminal carboxyl group forms a peptidic bond with the primary amine of the l-Lys side chain. In aqueous solution at room temperature, the (1)H NMR spectrum of pyoverdine PvdI-Ga(III) showed clear evidence of exchange broadening. At 253 K, two distinct conformations were observed and the measurement of structural constraints was possible. The three-dimensional structures of the two PvdI-Ga(III) conformers were determined, and analysis of the structures indicates that the observed conformational exchange involves a stereoisomerization of the metal binding coordination accompanied by a change in the global shape of the siderophore. This conformational transition was further characterized by heteronuclear relaxation experiments. The possible implications of this dynamic behavior for siderophore recognition by the receptor FpvAI are discussed.     

Inhibition of cadmium-induced oxidative injury in rat primary astrocytes by the addition of antioxidants and the reduction of intracellular calcium

Exposure of the brain to cadmium ions (Cd(2+)) is believed to lead to neurological disorders of the central nervous system (CNS). In this study, we tested the hypothesis that astrocytes, the major CNS-supporting cells, are resistant to Cd(2+)-induced injury compared with cortical neurons and microglia (CNS macrophages). However, treatment with CdCl(2) for 24 h at concentrations higher than 20 microM substantially induced astrocytic cytotoxicity, which also resulted from long-term exposure to 5 microM of CdCl(2). Intracellular calcium levels were found to rapidly increase after the addition of CdCl(2) into astrocytes, which led to a rise in reactive oxygen species (ROS) and to mitochondrial impairment. In accordance, preexposure to the extracellular calcium chelator EGTA effectively reduced ROS production and increased survival of Cd(2+)-treated astrocytes. Adenovirus-mediated transfer of superoxide dismutase (SOD) or glutathione peroxidase (GPx) genes increased survival of Cd(2+)-exposed astrocytes. In addition, increased ROS generation and astrocytic cell death due to Cd(2+) exposure was inhibited when astrocytes were treated with the polyphenolic compound ellagic acid (EA). Taken together, Cd(2+)-induced astrocytic cell death resulted from disrupted calcium homeostasis and an increase in ROS. Moreover, our findings demonstrate that enhancement of the activity of intracellular antioxidant enzymes and supplementation with a phenolic compound, a natural antioxidant, improves survival of Cd(2+)-primed astrocytes. This information provides a useful approach for treating Cd(2+)-induced CNS neurological disorders.     

Structural requirements for the tissue-specific and tissue-general functions of the Caenorhabditis elegans epidermal growth factor LIN-3.

Caenorhabditis elegans lin-3 encodes a homolog of the epidermal growth factor (EGF) family of growth factors. LIN-3 is the inductive signal for hermaphrodite vulval differentiation, and it is required for animal viability, hermaphrodite fertility, and the specification of anterior cell fates in the male B cell lineage. We describe the cloning of a lin-3 homolog from C. briggsae, sequence comparison of C. elegans lin-3 with C. briggsae lin-3, and the determination of molecular lesions in alleles of C. elegans lin-3, including three new alleles. We also analyzed the severity of phenotypes caused by the new and existing alleles of lin-3. Correlation of mutant phenotypes and their molecular lesions, as well as sequence comparison between two species, reveal that the EGF motif and the N-terminal portion of the cytoplasmic domain are important for the functions of LIN-3 in all tissues, while the C-terminal portion of the cytoplasmic domain is involved in the tissue-specific functions of lin-3. We discuss how the structure of lin-3 contributes to its functions in multiple developmental processes.     

The Metabolism of Hormones during Seed Germination and Dormancy: II. The Metabolism of 8-C-Zeatin in Bean Axes

8-¹⁴C-Zeatin is taken up rapidly and is extensively metabolized by excised bean axes during a 12-hour incubation at 26 C. Most of the radioactivity is found in the 80% ethanol soluble fraction and consists of zeatin, zeatin riboside, zeatin-5′-ribotide, as well as corresponding dihydrozeatin derivatives. The characterization of ¹⁴C-dihydrozeatin included crystallization to constant specific radioactivity. No cleavage of the zeatin side chain to adenine, hypoxanthine, their ribosides, or glycylpurine was detected. Dihydrozeatin has been previously isolated from yellow lupin seeds, and our experiments indicate that it can be derived through reduction of the side chain from preexisting cytokinin. While the total amount of zeatin metabolized is not affected by growth-inhibiting concentrations of abscisic acid or cycloheximide, the conversion to dihydrozeatin derivatives is curtailed. Although somewhat less effective than zeatin and zeatin riboside, dihydrozeatin and dihydrozeatin riboside also counteract the abscisic acid-induced growth inhibition.     

Hx-amides: DNA sequence recognition by the fluorescent Hx (p-anisylbenzimidazole)•pyrrole and Hx•imidazole pairings

Hx-amides are fluorescent hybrids of imidazole (I)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx–I/P–I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and ΔT_M studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T–A/T; Hx/IP prefers C–A/T; Hx/PI prefers A/T–C; and Hx/II prefers C–C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts.