Analysis of eosinophils and myeloid progenitor responses to modified forms of MPIF-2.

Myeloid progenitor inhibitory factor (MPIF)-2 is a beta-chemokine with select and potent activities on eosinophils and myeloid progenitors. In the beta-chemokine family, biological activity is modulated by differential processing of the amino-terminus. Here, for MPIF-2, we describe the biological activities of NH(2)-terminal deletion mutants and compare regions necessary for eosinophil and myeloid progenitor activities. Five MPIF-2 proteins with deletions at the amino-terminus were produced in Escherichia coli and assayed for calcium mobilization, chemotaxis and receptor binding activities on eosinophils, and for their ability to inhibit colony formation of human myeloid bone marrow progenitors. For eosinophils, deletion of the first two amino acids did not markedly alter activity, while subsequent truncations result in a complete loss of activity. One of the MPIF-2 mutants, MPIF-2 (P30-R99) was converted from an agonist to an antagonist of eotaxin, MPIF-2 and MCP-4 functional responses in eosinophil calcium flux and chemotaxis assays. Surprisingly, while displaying a complete loss of agonist activity toward eosinophils, MPIF-2 (P30-R99) retains ability to inhibit human bone marrow myeloid progenitor cell colony formation. In addition, processing at the amino terminus of MPIF-2 in vivo, may result in a chemokine with altered biological activities.

C-C chemokine receptor 3 antagonism by the beta-chemokine macrophage inflammatory protein 4, a property strongly enhanced by an amino-terminal alanine-methionine swap.

Allergic reactions are characterized by the infiltration of tissues by activated eosinophils, Th2 lymphocytes, and basophils. The beta-chemokine receptor CCR3, which recognizes the ligands eotaxin, eotaxin-2, monocyte chemotactic protein (MCP) 3, MCP4, and RANTES, plays a central role in this process, and antagonists to this receptor could have potential therapeutic use in the treatment of allergy. We describe here a potent and specific CCR3 antagonist, called Met-chemokine beta 7 (Ckbeta7), that prevents signaling through this receptor and, at concentrations as low as 1 nM, can block eosinophil chemotaxis induced by the most potent CCR3 ligands. Met-Ckbeta7 is a more potent CCR3 antagonist than Met- and aminooxypentane (AOP)-RANTES and, unlike these proteins, exhibits no partial agonist activity and is highly specific for CCR3. Thus, this antagonist may be of use in ameliorating leukocyte infiltration associated with allergic inflammation. Met-Ckbeta7 is a modified form of the beta-chemokine macrophage inflammatory protein (MIP) 4 (alternatively called pulmonary and activation-regulated chemokine (PARC), alternative macrophage activation-associated C-C chemokine (AMAC) 1, or dendritic cell-derived C-C chemokine (DCCK) 1). Surprisingly, the unmodified MIP4 protein, which is known to act as a T cell chemoattractant, also exhibits this CCR3 antagonistic activity, although to a lesser extent than Met-Ckbeta7, but to a level that may be of physiological relevance. MIP4 may therefore use chemokine receptor agonism and antagonism to control leukocyte movement in vivo. The enhanced activity of Met-Ckbeta7 is due to the alteration of the extreme N-terminal residue from an alanine to a methionine.

Recent trends in protein biochip technology.

This article presents current trends and advances in protein biochip technologies that rely upon extraction and retention of target proteins from liquid media. Analytical strengths as well as technical challenges for these evolving platforms are presented with particular emphasis on selectivity, sensitivity, throughput and utility in the post-genome era. A general review of protein biochip technology is provided, which delineates approaches for protein biochip format and operation, as well as protein detection. A focused discussion of three protein biochip technologies, Biomolecular Interaction Analysis (Biacore, Uppsala, Sweden), Surface Enhanced Laser Desorption/Ionisation (SELDI) ProteinChip Arrays (Ciphergen Biosystems, Fremont, CA, USA) and Fluorescent Planar Wave Guide (Zeptosens, Witterswil, Switzerland), follows along with examples of relevant applications.

Effects of increased in vivo excursion on digital range of motion and tendon strength following flexor tendon repair.

Postoperative rehabilitation is an important factor in determining functional outcome following intrasynovial flexor tendon repair. We hypothesized that a rehabilitation protocol that produced increased in vivo excursion would lead to increased digital range of motion and tendon strength and decreased adhesion formation in a canine model. Ninety-six flexor digitorum profundus tendons from 48 dogs were cut transversely and repaired by a multistrand suture technique. Postoperative rehabilitation was performed daily with a low excursion-low force (1.7-mm average excursion; < 10 N force) or a high excursion-low force (3.6 mm excursion; < 10 N force) protocol. After death of the dogs at 10, 21, or 42 days, specimens were evaluated for digital range of motion, tensile mechanical properties, elongation of the repair site, and adhesion formation. Our data indicate that the range of motion of digits whose tendons were at low or high excursion was similar to that of controls. Increased in vivo tendon excursion due to synergistic wrist motion did not significantly affect ex vivo flexion of the distal and proximal interphalangeal joints or tendon displacement (p > 0.05). Similarly, tensile properties (ultimate load, repair site rigidity, and repair site strain at 20 N and at failure) and length of the gap at the repair site were not significantly affected by increased excursion (p > 0.05). Severity of adhesion formation was reduced slightly by increased excursion (p = 0.04). Our findings indicate that 1.7 mm of tendon excursion is sufficient to prevent adhesion formation following sharp transection of the canine flexor tendon and that additional excursion provides little added benefit.

Detection of antibodies to the nonstructural 3C proteinase of hepatitis A virus.

Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and nonstructural proteins of the virus. However, vaccination with an inactivated vaccine produces antibodies exclusively to the structural proteins. Current diagnostic assays, such as the Abbott HAVAB test, used to determine exposure to HAV detect antibodies only to the structural proteins and as a result are not able to distinguish between a natural infection and vaccination with an inactivated virus. Therefore, an ELISA was developed that is specific for antibodies to the nonstructural protein 3C of HAV and thus serves to document the occurrence of viral replication. Antibodies to the proteinase were not detected by this assay in serum from HAVAB-seropositive primates that were immunized with inactivated HAV. However, antibodies to the proteinase were detected in the serum of all primates experimentally infected with virulent HAV and in the serum of naturally infected humans.

In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.

Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.

Parent and family support groups with African American families: the process of family and community empowerment.

This article describes a process of family and community empowerment in which psychologists, along with community, school and religious leaders, intervened on a multisystemic level and formed a parent and family support group to empower families in helping their at-risk adolescents to succeed. The adolescents, who were predominantly African American, had been arrested for fighting at school and were experiencing academic and behavioral difficulties. Critical incidents in the group development and the family and community empowerment process are described.

Occipital condylar dysplasia in two Jacob sheep.

Two young Jacob sheep which presented with severe ataxia and torticollis had abnormally formed atlanto-occipital joints. Postmortem examination revealed marked dissimilarity in size between the left and right occipital condyles, with reduction in size of the foramen magnum. The atlantoaxial joint and dens were normally formed, and were not abnormally positioned in radiographs taken of one lamb. Histological evaluation of the cervicomedullary junction demonstrated extensive loss of axons and myelin, gliosis, and mild hydromyelia in one lamb.