Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in gene rich bands

The inactive X chromosome (Xi) forms a heterochromatic structure in the nucleus that is known to have several modifications to specific histones involving acetylation or methylation. Using three different antibodies in four different cell lines, we demonstrate that the Xi in human and mouse cells is highly enriched in ubiquitinated protein(s), much of which is polyubiquitinated. This ubiquitination appears specific for the Xi as it was not observed for centromeres or other regions of heterochromatin. Results using an antibody specific to ubiquitinated H2A provide a clear link between H2A ubiquitination and gene repression, as visualized across an entire inactive chromosome. Interestingly, the ubiquitination of the chromosome persists into mitosis and can be seen in a reproducible banded pattern. This pattern matches that of Xist RNA which forms bands as it detaches from the mitotic X chromosome. Both ubiquitination and Xist RNA appear enriched in gene dense regions and depleted in gene poor bands, but do not correlate with L1 LINE elements which have been suggested as key to X-inactivation. These results provide evidence that ubiquitination along with Xist RNA plays an important role in the formation of facultative heterochromatin during X-inactivation.     

Larval morphology, genetic divergence, and contrasting levels of host manipulation between forms of Pomphorhynchus laevis (Acanthocephala)

Studies on parasite species with a wide geographic and ecological range may be confounded by still equivocal taxonomic identification. Here, we investigated genetic polymorphism and behavioural changes induced in a common intermediate host, in two different forms of Pomphorhynchus laevis based on the morphology of the larval infective stage (cystacanth). A 'smooth type' (S) and a 'wrinkled type' (W) of cystacanth were distinguished based on their surface and shape. We analysed sequence divergence at both nuclear (ribosomal gene 18S rDNA, and ribosomal internal transcribed spacers, ITS1/ITS2) and mitochondrial (cytochrome c oxidase subunit 1) genes of P. laevis cystacanths and adults at various geographical scales. A high level of sequence divergence at ITS1, ITS2 and cytochrome c oxidase subunit 1 (11, 8 and 20%, respectively) was found between these two forms. The divergence pattern consistently discriminated two groups independently of geographic origin or host, and was congruent with larval morphology. The two forms also strongly differed in the intensity of behavioural change induced in their common intermediate host, Gammarus pulex, with the S-type parasite inducing a positive phototactism, whereas W-type infected gammarids were as photophylic as uninfected ones. Overall, our data strongly support the specific status of these two forms. We suggest that smooth cystacanths correspond to P. laevis, whereas wrinkled cystacanths might correspond to the previously described and poorly documented, Pomphorhynchus tereticollis, considered a synonym of P. laevis. This study also confirms the value of a joint analysis of internal transcribed spacers and cytochrome c oxidase subunit 1 genes to biogeographic studies on these species. Finally, we emphasize the importance of linking morphological and biological characteristics of acanthocephalan cystacanths to molecular data, in the study of the evolutionary ecology and systematics of this group.     

Esterase catalysis of substrate vapour: enzyme activity occurs at very low hydration

It has been generally accepted that enzyme activity requires a minimal hydration of about 0.2 g H2O g(-1) protein. This fits well with evidence that hydration above this level is associated with the onset of intramolecular motions. The influence of enzyme hydration on the hydrolysis of substrate by Candida rugosa Lipase B and pig liver esterase was investigated. Each enzyme was studied as a powder at various hydration levels, using vapour phase ethyl butyrate as substrate. This procedure allows the separation of those effects that are due to hydration from those arising from diffusional constraints. We found hydrolytic activity in both enzymes at all hydration levels above zero (between 0.054-0.47 and 0.029-0.60 g H2O g(-1) protein, respectively) that were investigated. The lowest hydration level investigated, <0.03 g H2O g(-1) enzyme, corresponded to a water/enzyme mole ratio of 100 and a coverage of about 10% of the enzyme surface by water molecules. The hydrolytic activity of both enzymes was dependent on protein hydration. However, since the hydrolysis of ethyl butyrate requires water as a second substrate, the absence of activity at zero hydration does not rule out the possibility of enzyme activity in the absence of water. These results suggest that the properties conferred on proteins by water, at least above 10% surface coverage (in this case corresponding to a hydration level of 0.03 g H2O g(-1) protein), are not a requirement for enzyme catalysis.     

Crystal structure of the apo forms of psi 55 tRNA pseudouridine synthase from Mycobacterium tuberculosis: a hinge at the base of the catalytic cleft

The three-dimensional structure of the RNA-modifying enzyme, psi55 tRNA pseudouridine synthase from Mycobacterium tuberculosis, is reported. The 1.9-A resolution crystal structure reveals the enzyme, free of substrate, in two distinct conformations. The structure depicts an interesting mode of protein flexibility involving a hinged bending in the central beta-sheet of the catalytic module. Key parts of the active site cleft are also found to be disordered in the substrate-free form of the enzyme. The hinge bending appears to act as a clamp to position the substrate. Our structural data furthers the previously proposed mechanism of tRNA recognition. The present crystal structure emphasizes the significant role that protein dynamics must play in tRNA recognition, base flipping, and modification.     

A primate-dominant third glycosylation site of the beta2-adrenergic receptor routes receptors to degradation during agonist regulation

beta(2)-adrenergic receptors (beta(2)AR) of all species are N-linked glycosylated at amino terminus residues approximately 6 and approximately 15. However, the human beta(2)AR has a potential third N-glycosylation site at ECL2 residue 187. To determine whether this residue is glycosylated and to ascertain function, all possible single/multiple Asn --> Gln mutations were made in the human beta(2) AR at positions 6, 15, and 187 and were expressed in Chinese hamster fibroblast cells. Substitution of Asn-187 alone or with Asn-6 or Asn-15 decreased the apparent molecular mass of the receptor on SDS-PAGE in a manner consistent with Asn-187 glycosylation. All receptors bound the agonist isoproterenol and functionally coupled to adenylyl cyclase. However, receptors without 187 glycosylation failed to display long term agonist-promoted down-regulation. In contrast, loss of Asn-6/Asn-15 glycosylation did not alter down-regulation. Cell surface distribution and agonist-promoted internalization of receptors and recruitment of beta-arrestin 2 were unaffected by the loss of 187 glycosylation. Furthermore, acutely internalized wild-type and Gln-187 receptors were both localized by confocal microscopy to early endosomes. During prolonged agonist exposure, wild-type beta(2)AR co-localized with lysosomes, consistent with trafficking to a degradation compartment. However, Gln-187 beta(2)AR failed to co-localize with lysosomes despite agonist treatments up to 18 h. Phylogenetic analysis revealed that this third glycosylation site is found in humans and other higher order primates but not in lower order primates such as the monkey. Nor is this third site found in rodents, which are frequently utilized as animal models. These data thus reveal a previously unrecognized beta(2)AR regulatory motif that appeared late in primate evolution and serves to direct internalized receptors to lysosomal degradation during long term agonist exposure.     

Photoreceptor cGMP phosphodiesterase delta subunit (PDEdelta) functions as a prenyl-binding protein

Bovine PDEdelta was originally copurified with rod cGMP phosphodiesterase (PDE) and shown to interact with prenylated, carboxymethylated C-terminal Cys residues. Other studies showed that PDEdelta can interact with several small GTPases including Rab13, Ras, Rap, and Rho6, all of which are prenylated, as well as the N-terminal portion of retinitis pigmentosa GTPase regulator and Arl2/Arl3, which are not prenylated. We show by immunocytochemistry with a PDEdelta-specific antibody that PDEdelta is present in rods and cones. We find by yeast two-hybrid screening with a PDEdelta bait that it can interact with farnesylated rhodopsin kinase (GRK1) and that prenylation is essential for this interaction. In vitro binding assays indicate that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 co-precipitate with a glutathione S-transferase-PDEdelta fusion protein. Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of PDEdelta and dansylated prenyl cysteines as fluorescent ligands, we show that PDEdelta specifically binds geranylgeranyl and farnesyl moieties with a Kd of 19.06 and 0.70 microm, respectively. Our experiments establish that PDEdelta functions as a prenyl-binding protein interacting with multiple prenylated proteins.     

The burden of genetic disease on inpatient care in a children's hospital

The important role of genetics in pediatric illness has been increasingly recognized, but the true impact has not been well delineated. An important study of pediatric inpatient admissions to a children's hospital in 1978 found a genetic basis for disease in just less than half of admitted patients. We sought to update this study in light of current hospitalization practices and new knowledge about genetics. We systematically reviewed the records of 5,747 consecutive admissions (4,224 individuals), representing 98% of patients admitted in 1996 to Rainbow Babies and Children's Hospital (Cleveland, OH). Each patient was assigned to one of five groups on the basis of the presence or absence of an underlying chronic medical condition and whether that condition had a genetic basis or susceptibility. An underlying disorder with a significant genetic component was found in 71% of admitted children. The vast majority (96%) of underlying chronic disorders in children in this study were either clearly genetic or had a genetic susceptibility. Total charges for 1996 were >$62 million, of which $50 million (81%) was accounted for by disorders with a genetic determinant. The 34% of admissions with clearly genetic underlying disorders accounted for 50% (>$31 million) of the total hospital charges. The mean length of stay was 40% longer for individuals with an underlying disease with a genetic basis than for those with no underlying disease. Charges and length of stay were similar for children with underlying chronic disorders, regardless of the cause. This study begins to quantify the enormous impact of genetic disease on inpatient pediatrics and the health care system. Additional study and frank public discourse are needed to understand the implications on the future health care workforce and on the utilization of health care resources.     

Regulated expression of the receptor-like tyrosine phosphatase CD148 on hemopoietic cells

CD148 is a receptor-like protein tyrosine phosphatase expressed on a wide variety of cell types. Through the use flow cytometry and immunofluorescence microscopy on tissue sections, we examined the expression of CD148 on multiple murine hemopoietic cell lineages. We found that CD148 is moderately expressed during all stages of B cell development in the bone marrow, as well as peripheral mature B cells. In contrast, CD148 expression on thymocytes and mature T cells is substantially lower. However, stimulation of peripheral T cells through the TCR leads to an increase of CD148 expression. This up-regulation on T cells can be partially inhibited by reagents that block the activity of src family kinases, calcineurin, MEK, or PI3K. Interestingly, CD148 levels are elevated on freshly isolated T cells from MRL lpr/lpr and CTLA-4-deficient mice, two murine models of autoimmunity. Together, these expression data along with previous biochemical data suggest that CD148 may play an important regulatory role to control an immune response.     

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.