MCA/MR syndrome in two female siblings: new entity or variant examples of Coffin-Lowry versus Atkin-Flaitz syndromes?

We report two sisters with mental retardation, coarse facial features, telecanthus, flat malar region, prominent lower lip, kyphoscoliosis, and tapering fingers. Although these patients' phenotypes showed considerable overlap with the Coffin-Lowry and the Atkin-Flaitz syndromes, their overall picture makes these diagnoses controversial.     

Slow conformational exchange in DNA minihairpin loops: A conformationalstudy of the circular dumbbell d〈pCGC-TT-GCG-TT〉

In recent years various examples of highly stable two-residue hairpin loops (miniloops) in DNA have been encountered. As the detailed structure and stability of miniloops appear to be determined not only by the nature and sequence of the two bases in the loop, but also by the closing base pair, it is desirable to carry out in-depth studies of especially designed small model DNA compounds. Therefore, a circular DNA dumbbell-like molecule is tailored to consist of a stem of three Watson–Crick base pairs, flanked on each side by a minihairpin loop. The resulting circular DNA decanter 5′-d〈pCGC- TT-GCG- TT〉 -3′ ( I ) is studied in solution by means of nmr spectroscope. At a temperature of 269 K the molecule occurs in a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4). L2L2 contains three Watson–Crick C-G base pairs and two two-residue loops (H2-family type) in opposite parts of the molecule. On raising the temperature from 269 to 314 K. The L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted closing G-C base pair in the 5′-GTTC-3′ loop with only one remaining solvent-accessible hydrogen bond between NH_α of the cytosine C(1) and O6 of the guanine G(8), whereas the opposite 5′-CTTG-3′ loop remains stable. The disruption of the C(1)-G(8) base pair in the L2L4 form is correlated with the presence of a syn orientation for the C(1) base at the 5′-3′ loop-stem junction in the 5′-GTTC-3′ loop. The two conformers. L2L2 and L2L4, occur in slow equilibrium (2–20 s^?1). Moderate line broadening of specific ¹H, ¹³C, and ³¹P resonances of residues C(1), G(8), T(9), and T(10) at low temperatures, due to chemical exchange between L2L2 and L2L4, show that the interconversion from an anti to syn conformer in residue C(1) has a small local effect on the structure of the dumbbell. T₁ relaxation measurements, chemical-shift considerations, and complete hand-shape calculations of the exchange process of the G(8) imino proton reveal a possibility for the existence of multiconformational slates in the anti–syn equilibrium. © 1995 John Wiley & Sons, Inc.     

Gly118Asp is a SCA14 founder mutation in the Dutch ataxia population

Missense mutations in the PRKCG gene have recently been identified in spinocerebellar ataxia 14 (SCA14) patients; these include the Gly118Asp mutation that we found in a large Dutch autosomal dominant cerebellar ataxia (ADCA) family. We subsequently screened the current Dutch ataxia cohort (approximately 900 individuals) for SCA14 mutations in the Cys2 region of the PRKCG gene. We identified the Gly118Asp mutation in another eight individuals from five small families. Haplotype analysis identified a shared chromosomal region surrounding the SCA14 gene, and genealogical research was able to link all these ADCA patients to a single common ancestor. We therefore confirmed that the Gly118Asp mutation is a SCA14 founder mutation in the Dutch ADCA population.     

Probing solvent accessibility of transthyretin amyloid by solution NMR spectroscopy

The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six beta-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F' and H-H' with a prolongation of these beta-strands and, interestingly, with a possible shift in beta-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented.     

Structure of the Tyrosine-sulfated C5a Receptor N Terminus in Complex with Chemotaxis Inhibitory Protein of Staphylococcus aureus

Complement component C5a is a potent pro-inflammatory agent inducing chemotaxis of leukocytes toward sites of infection and injury. C5a mediates its effects via its G protein-coupled C5a receptor (C5aR). Although under normal conditions highly beneficial, excessive levels of C5a can be deleterious to the host and have been related to numerous inflammatory diseases. A natural inhibitor of the C5aR is chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). CHIPS is a 121-residue protein excreted by S. aureus. It binds the N terminus of the C5aR (residues 1-35) with nanomolar affinity and thereby potently inhibits C5a-mediated responses in human leukocytes. Therefore, CHIPS provides a starting point for the development of new anti-inflammatory agents. Two O-sulfated tyrosine residues located at positions 11 and 14 within the C5aR N terminus play a critical role in recognition of C5a, but their role in CHIPS binding has not been established so far. By isothermal titration calorimetry, using synthetic Tyr-11- and Tyr-14-sulfated and non-sulfated C5aR N-terminal peptides, we demonstrate that the sulfate groups are essential for tight binding between the C5aR and CHIPS. In addition, the NMR structure of the complex of CHIPS and a sulfated C5aR N-terminal peptide reveals the precise binding motif as well as the distinct roles of sulfated tyrosine residues sY11 and sY14. These results provide a molecular framework for the design of novel CHIPS-based C5aR inhibitors.The human complement system is a key component of the innate host defense directed against invading pathogens. Complement component C5a is a 74-residue glycoprotein generated via complement activation by cleavage of the α-chain of its precursor C5. C5a is a strong chemoattractant involved in the recruitment of neutrophils and monocytes, activation of phagocytes, release of granule-based enzymes, and in the generation of oxidants (1, 2). C5a exerts its effect by activating the C5a receptor (C5aR).³ Although this is a highly efficient process, excessive or erroneous activation of the C5aR can have deleterious effects on host tissues. C5a has been implicated in the pathogenesis of many inflammatory and immunological diseases, including rheumatoid arthritis, inflammatory bowel disease, immune complex disease, and reperfusion injury (3, 4). Consequently, there is an active ongoing search for compounds that suppress C5a-mediated inflammatory responses.Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a 121-residue protein excreted by S. aureus, which efficiently inhibits the activation of neutrophils and monocytes by formylated peptides and C5a (5, 6). CHIPS specifically binds to the formylated peptide receptor (FPR) and the C5aR with nanomolar affinity (K_d = 35.4 ± 7.7 nm and 1.1 ± 0.2 nm, respectively) (7), thereby suppressing the inflammatory response of the host. A CHIPS fragment lacking residues 1-30 (designated CHIPS_31-121) has the same activity in blocking the C5aR compared with wild-type CHIPS (8). CHIPS_31-121 is a compact protein comprising an α-helix packed onto a four-stranded anti-parallel β-sheet (8). C5a has an entirely different fold (PDB ID code 1KJS) and is comprised of an anti-parallel bundle of four α-helices stabilized by three disulfide bonds (9, 10). Preliminary experiments indicated that CHIPS binds exclusively to the extracellular N-terminal portion of the C5aR (7). In contrast, the binding of C5a by its receptor involves two separate binding sites: C5a residues located in the region between 12-46 (11, 12) bind to a primary binding site partly coinciding with the binding site of CHIPS, while the C terminus of C5a (residues 69-74) binds to the activation domain of the C5aR located in the receptor core (13). Because of their dissimilarity in sequence and structure, the binding sites of CHIPS and C5a are not identical (11). The present working model is that CHIPS interferes with the primary binding site of C5a located at the N terminus of the C5aR, thereby preventing the C-terminal tail of C5a from contacting the activation domain of the C5aR and blocking downstream signaling. Currently, the development of C5aR inhibitors has been focused primarily on mimicking C5a in order to directly interrupt C5a-mediated C5aR signaling (3, 4, 14). Understanding the interactions between CHIPS and the C5aR may provide valuable insights toward the development of new C5aR antagonists.Postma et al. (15) proposed that residues involved in CHIPS binding are located between residues 10-18 of the C5aR. Specifically, the acidic residues Asp-10, Asp-15, and Asp-18 and residue Gly-12 appear to be critical for binding. High affinity binding was observed between ¹²⁵I-labeled CHIPS and the N-terminal portion of the C5aR (residues 1-38) expressed on the cell surface of HEK293 cells (K_d = 29.7 ± 4.4 nm). In contrast, very moderate affinity between CHIPS and a synthetic C5aR N-terminal peptide (residues 1-37; K_d = 40 ± 19 μm), measured by isothermal titration calorimetry (ITC), was recently reported by Wright et al. (16). The discrepancy in the magnitude of these dissociation constants may be explained by the presence of two sulfate groups on tyrosine 11 and 14 of the C5aR N terminus expressed on the cell surface of HEK293 cells, which are absent in the synthetic C5aR peptide utilized by Wright et al. (16). Farzan et al. (17) stressed the critical role of these sulfate groups in activation of the C5aR by C5a. Previous mutational studies employing FITC-labeled CHIPS, however, suggested that the sulfate groups had only a limited effect on the binding affinity (15).To resolve these discrepancies, we set out to chemically synthesize several sulfated and unsulfated peptides representing the N terminus of the human C5aR. We have measured the binding affinities of these peptides to CHIPS_31-121 by ITC and used the C5aR peptide with the highest affinity to determine the structure of the complex between CHIPS_31-121 and the C5aR N terminus by NMR spectroscopy.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

NMR characterization of a 264-residue hyperthermostable endo-β-1,3-glucanase

Insight into the hyperthermostable endo-β-1,3-glucanase pfLamA from Pyrococcus furiosus is obtained by using NMR spectroscopy. pfLamA functions optimally at 104 °C and recently the X-ray structure of pfLamA has been obtained at 20 °C, a temperature at which the enzyme is inactive. In this study, near-complete (>99%) NMR assignments are presented of chemical shifts of pfLamA in presence and absence of calcium at 62 °C, a temperature at which the enzyme is biologically active. The protein contains calcium and the effects of calcium on the protein are assessed. Calcium binding results in relatively small chemical shift changes in a region distant from the active site of pfLamA and thus causes only minor conformational modifications. Removal of calcium does not significantly alter the denaturation temperature of pfLamA, implying that calcium does not stabilize the enzyme against global unfolding. The data obtained form the basis for elucidation of the molecular origins involved in conformational stability and biological activity of hyperthermophilic endo-β-1,3-glucanases at extreme temperatures.