Cloning and sequencing of hen magnum cDNAs encoding vitelline membrane outer layer protein I (VMO-I).

Two cDNAs encoding hen vitelline membrane outer layer protein I (VMO-I), which is classified as a new type of multi-beta-sheet assembly, were cloned and sequenced. Northern blot analysis using vmo-I cDNA as a probe showed the presence of three mRNA species. Strikingly, expression of these mRNAs was restricted to a specific region of the hen oviduct, the area joining the infundibulum to the magnum.

Construction of a divalent cell adhesive lysozyme by introducing the Arg-Gly-Asp sequence at two sites.

To increase the cell adhesion activity of 74RGD4, an RGDS-inserted mutant between Val74 and Asn75 of human lysozyme, one more site for the RGD introduction was investigated in the lysozyme molecule. We found that 47RGD4 with RGDS in place of AGDR (residues 47 to 50) in a beta-turn region possesses the same level of adhesion activity as that of 74RGD4. The acceptance of the RGD introduction in the beta-turn region of human lysozyme is in good agreement with recent studies on the functional conformation of RGD. We constructed (47,74)RGD4, a mutant containing RGD at two sites, by combining the N-terminal domain of 47RGD4 and the C-terminal domain of 74RGD4. The (47,74)RGD4 lysozyme, with two functional RGD sequences, exhibits even higher cell adhesion activity than that of 74RGD4 or 47RGD4.

Neurocrescin: a novel neurite-outgrowth factor secreted by muscle after denervation.

Regeneration of injured axons at neuromuscular junctions has been assumed to be regulated by extracellular factors that promote neurite outgrowth. We report here the cloning of a novel neurite outgrowth factor, designated neurocrescin, from chick denervated skeletal muscle. A recombinant neurocrescin promoted neurite outgrowth from cultured neurons of spinal cord and telencephalon of chick embryo. It was expressed predominantly in neural tissue and muscle, and was secreted extracellularly after intramolecular cleavage. This truncated form was detected in denervated muscle but not in innervated muscle. Thus, neurocrescin appears to be a novel neurite outgrowth factor that is secreted in an activity-dependent fashion. A highly homologous counterpart was also cloned from mouse brain.

Chick muscle-derived protein 62: a novel neurite outgrowth promoting protein.

A 3.2 kb chick cDNA clone that coded for a novel muscle-derived protein, MDP62, was isolated from a cDNA library of the denervated crus muscles using an antibody which inhibited the neurite (dendritic and axonal processes) outgrowth activity. MDP62 consisted of 539 aa with a calculated molecular mass of 62 k. The predicted protein sequence was hydrophilic and exhibited an extended coiled-coil domain and a leucine zipper motif. A recombinant protein promoted the neurite outgrowth from the cultured chick neurons of the telencephalon in a dose dependent manner. Northern blotting revealed that MDP77 was ubiquitously expressed. In the transfected COS-7 cells with the cDNA of the epitope-tagged MDP62, the expressed protein was detected in the culture medium, suggesting that the MDP62 might be secreted.

Children with chronic conditions in a pediatric emergency department.

The objective of this study was to describe the use of a pediatric emergency department (PED) by children with chronic conditions. The study design was retrospective and descriptive in an urban tertiary care pediatric hospital setting. We reviewed 8561 visits to a PED over a three-month time period. Two thousand twenty-four (24%) of the visits were by children with one or more chronic conditions. There were no interventions. The mean age of the patients was 4.9 years, and 61% were male. Thirty-one percent of the patients sought care between 8 AM and 5 PM Monday through Friday. Five subspecialty areas accounted for 86% of the chronic conditions seen: asthma (43%), neurology (15%), hematology/oncology (14%), neurosurgery (10%), and cardiology (4%). Twenty-eight percent of the chronically ill patients were admitted as compared to 11% of the nonchronically ill patients (P < 0.001). One percent of the chronically ill patients were admitted to the intensive care unit as compared to 0.03% of the nonchronically ill patients (P < 0.0001). It was concluded that children with chronic conditions account for one-quarter of all PED visits. Sixty-nine percent of those visits were made during evening/ nighttime hours or on the weekend. A relatively large percentage of these children were admitted. The pediatric emergency physicians provide an important service to both the children with chronic conditions and the subspecialists who care for them. PEDs may need to refine emergency department systems to serve this group of patients as efficiently and effectively as possible.

Functional analysis and modeling of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme.

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we have constructed mutant proteins by inserting RGD-containing sequences flanked by two Cys residues between Val74 and Asn75 of human lysozyme. CRGDC-, CRGDSC-, and CGRGDSC-inserted mutant lysozymes were expressed in yeast, purified, and designated as Cys-RGD3, Cys-RGD4, and Cys-RGD5, respectively. In baby hamster kidney cells, these mutants were shown to possess high cell adhesion activity by interaction with vitronectin receptor (integrin alpha v beta 3), and this activity is 2-3-fold higher than that of the RGDS-inserted mutant lysozyme, RGD4. The mutant proteins also inhibited the binding of human fibrinogen to its receptor (integrin alpha IIb beta 3) at a lower concentration than the RGD4 protein. Peptide mapping and mass spectrometric analyses showed that the two inserted Cys residues in these mutants are linked to each other without any effects on the mode of the four disulfide bonds present in native human lysozyme. These results suggest that the introduction of a conformational constraint into the RGD region significantly increases the cell adhesion activity. The conformation of the RGD region in Cys-RGD4 was modeled by a Monte Carlo simulation. Most of the sampled conformations were grouped into three classes; the first is characterized by an extended Gly conformation, the second assumes a type II' beta turn, and the third has a salt bridge between Arg and Asp.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of recombinant cDNA clones for canine cardiac phospholamban.

Complementary DNA clones specific for phospholamban have been isolated from a canine cardiac cDNA library. The amino acid sequence deduced from the cDNA sequence showed that phospholamban consisted of 52 amino acid residues and was synthesized without an amino-terminal signal sequence. The RNA blot analysis revealed that phospholamban mRNAs were represented by two main species of approximately 1.2kb and approximately 2.8kb. These mRNAs appeared to differ primarily in the length of the 3' untranslated region.

O-glycosylation of the Thr70 residue of cell-adhesive lysozyme in yeast.

The cell-adhesive protein Cys-RGD4 has been constructed using a yeast expression system by inserting the sequence Cys-Arg-Gly-Asp-Ser-Cys (CRGDSC) between Val74 and Asn75 of human lysozyme [Yamada, T., Uyeda, A., Kidera, A. & Kikuchi, M. (1994b) Biochemistry 33, 11678-11683]. The Cys74a, Arg74b, Gly74c, Asp74d, Ser74e, Cys74f-lysozyme mutant, purified from the yeast culture supernatant contained glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the Thr70 residue in the Cys-RGD4 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of two hexose residues in the major variant, and one, three, four, or five hexose residues in the minor variants. All of these hexose residues were identified as mannose by analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants. No other glycosylation was observed, although the Cys-RGD4 molecule possesses a total of 12 threonine and serine residues. In addition, the Thr70 residue is not glycosylated in either native lysozyme or the Arg-Gly-Asp-Ser (RGDS)-inserted mutant, RGD4 [Yamada, T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda, A., Maeda, T., Titani, K., Sekiguchi, K. & Kikuchi, M. (1993) J. Biol. Chem. 268, 10588-10592]. Thus, this O-glycosylation seems to be specific for both the mutant lysozyme molecule and the site of the threonine residue. Structural analyses of these lysozymes by X-ray crystallography suggest that the conformation of the serine-containing or threonine-containing region can affect the specificity of yeast O-glycosylation.

Injury prevention education using pictorial information.

BACKGROUND: Written materials used in pediatric public health settings often exceed the reading skills of caretakers. OBJECTIVE: To compare a pictorial anticipatory guidance (PAG) sheet requiring limited reading skills to a TIPP (The Injury Prevention Program) sheet for providing injury prevention information to low-income urban families. DESIGN AND SETTING: A convenience sample of families with children treated at an urban pediatric clinic affiliated with a teaching hospital. Methods. Parents of children

MDP77: A novel neurite-outgrowth-promoting protein predominantly expressed in chick muscles.

A 4.7 kb chick cDNA clone that coded for the novel muscle-derived protein, MDP77, was isolated from a cDNA library of the denervated crus muscles using an antibody which inhibited the neurite outgrowth activity. MDP77 consisted of 676 aa with a calculated molecular mass of 77 k. The deduced amino acid sequence exhibited an extended coiled-coil domain and a leucine zipper motif. A recombinant protein promoted the neurite-outgrowth from the cultured chick neurons of the spinal cord in a dose-dependent manner. Northern blotting and in situ hybridization revealed that MDP77 was predominantly expressed in the cardiac and the skeletal muscles. In the COS-7 cells transfected with the cDNA of the epitope-tagged MDP77, the expressed protein was detected in the culture medium, suggesting that the MDP77 was secreted.

HSP90 has neurite-promoting activity in vitro for telencephalic and spinal neurons of chick embryos.

We purified a protein from the extract of denervated chick muscle. The protein had neurite-promoting activity in vitro for the chick telencephalic neurons and spinal neurons. Partial amino acid sequencing and immunoblotting revealed that the protein was chick heat shock protein 90 (HSP90). Commercially available bovine HSP90 and recombinant chick HSP90 expressed in Escherichia coli also showed the same activity. Since HSP10 (GroES) and HSP60 (GroEL) exhibited no activity for neurite outgrowth in the same culture, this activity was specific to HSP90 among the heat shock proteins.

Identification of active regions for neurite outgrowth activity of neurocrescin.

We previously identified and cloned a neurite outgrowth promoting protein, Neurocrescin (NC), from the extract of the chick denervated leg muscles. In this study, we explored the active region of NC for neurite outgrowth. Using the deletion mutants of NC, we tested their neurite outgrowth activity in the cultured telencephalic neurons of E5 chick embryos. We found three regions which independently had significant neurite outgrowth activity comparable with that of the extract of the chick denervated leg muscles. These regions were not homologous to any well-known active sites such as the laminin active region, IKVAV. In parallel, searching the endogenous deletion mutants of NC in the rat brain, we cloned a mutant in which the region including the larger part of one of the three active regions was deleted. The neurite outgrowth activity of the mutant was significantly lower than that of normal NC. These results suggest the physiological significance of these active regions.

Site-specific O-glycosylation of cell adhesive lysozyme in yeast.

The cell adhesive protein RGD8 has been constructed using a yeast expression system by inserting eight amino acid residues (TGRGDSPA) between Val74 and Asn75 of human lysozyme [Yamada et al. (1993) J. Biol. Chem. 268, 10588-10592]. Purified RGD8 from yeast culture supernatant was found to contain glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the inserted Thr residue in the RGD8 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of four or five hexose residues in the glycosylated variants. Only mannose was detected in the sugar analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants, and the structures of these carbohydrate chains were identified as Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha and Man alpha 1-3Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha by 1H-NMR spectroscopy. No other glycosylation was found, although the RGD8 molecule possesses a total of 13 Thr and Ser residues. In addition, no O-glycosylation was observed when the RGD8 protein was expressed in mouse L-cells. Thus, this O-glycosylation looks specific for yeast and the site of the Thr residue. The O-glycosylated variants of RGD8 exhibited a high level of adhesion activity to baby hamster kidney cells, which was almost comparable to that of the unglycosylated form.

Structural and functional analyses of the Arg-Gly-Asp sequence introduced into human lysozyme.

To determine the functional conformation of the Arg-Gly-Asp (RGD) sequence, we have constructed mutant proteins by inserting 4-12 amino acid residues from the RGD region of human fibronectin between Val74 and Asn75 of human lysozyme. RGDS-, GRGDSP-, TGRGDSPA-, VTGRGDSPAS-, and AVTGRGDS-PASS-introduced mutant lysozymes were expressed in yeast, purified, and designated as RGD4, -6, -8, -10, and -12, respectively. Using baby hamster kidney cells, RGD8, RGD10, and RGD12 were shown to possess high cell adhesion activity nearly equal to 10% of human vitronectin activity. RGD4 and RGD6 exhibited somewhat lower cell adhesion activity. The activities of these mutant proteins were inhibited by the addition of either GRGDSP peptide or polyclonal antibody against vitronectin receptor, as was the case for the vitronectin activity. The results suggest that the cell adhesion signals are transduced to cells through the interaction with the vitronectin receptor. The three-dimensional structures of RGD4 and RGD8 were determined at 1.8-A resolution by x-ray crystallography. A model of the inserted region in RGD4 could be built in the electron density map, but the positions of the preceding residues, Ala73-Val74, were uncertain. The inserted region in RGD8 did not demonstrate continuous electron densities. The results suggest that these RGD sequence-containing regions are highly flexible and that such flexibility could allow the conformation of the RGD regions to be induced to fit into the binding pocket of the integrin receptor.

Seroepidemiologic survey of captive Old-World primates for antibodies to human and simian retroviruses, and isolation of a lentivirus from sooty mangabeys (Cercocebus atys).

Sera from 526 Old-World monkeys and apes, representing 50 species and 20 genera and living in US zoos and vivaria, were screened for antibodies to HTLV-I, HTLV-III/LAV, and simian-AIDS retrovirus, type I (SRV-I). Sera were screened initially by ELISA, and ELISA-positive sera, as well as ELISA-negative sera from cage contacts, were further tested by Western blotting. A large number of false-positive and a small number of false-negative ELISA sera were identified. Although most true positive reactions were directed to a single retrovirus, a number of individuals from 4 species were positive for more than one retrovirus. Specific seroreactivity to HTLV-I was found in 39/526 (7%) animals of 15 species. True positive reactions to SRV-I were found in 21/516 (4%) animals, including talapoins and 2 species of macaques. Specific serologic reactions to HTLV-III/LAV were detected in 23/526 (4%) monkeys. Many of the HTLV-III/LAV seropositive animals were from one mixed-species zoo exhibit, containing sooty mangabeys, mandrills, Kolb's guenons, and talapoins. A type D virus was isolated from the blood of 3/10 SRV-I antibody-positive Tonkeana macaques, but from none of 11 seropositive talapoins. A lentivirus was isolated from the blood of 4/7 HTLV-III/LAV seropositive sooty mangabeys, but not from seropositive talapoins in the same exhibit or from 2 seropositive colobus from another zoo. The sooty mangabey lentivirus produced generalized lymphadenopathy, leukopenia, and decreased levels of T4 lymphocytes in 2 experimentally infected rhesus macaques.

Evidence for a lentiviral etiology in an epizootic of immune deficiency and lymphoma in stump-tailed macaques (Macaca arctoides).

A retrospective study determined that an epizootic of immune suppression and lymphoma in stump-tailed macaques (Macaca arctoides) that began in 1976 was associated with a horizontally spread lentivirus infection. This conclusion was based on serology, epidemiology, pathology, and virus isolation. The lesions found in the stump-tailed macaques were more compatible with lesions seen in SIV-infected rhesus than those seen in rhesus macaques infected with type D retroviruses. A lentivirus, isolated from a rhesus inoculated with lymph node homogenate from a stump-tailed macaque, was designed SIVstm and was pathogenic for rhesus macaques. The isolate was antigenically related to other SIVs as well as to HIV-1 and HIV-2. Two surviving stump-tailed macaques sent to another colony carried SIVstm latently for at least 7 years and disseminated it throughout that colony.