Activation of extracellular matrix metalloproteinases in equine laminitis

Samples of connective tissue obtained from the hoof of six laminitic and eight non-laminitic adult horses were analysed zymographically to investigate whether connective tissue matrix metalloproteinases are activated or induced during laminitis. The activity or matrix metalloproteinases was substantially greater in the tissues from the laminitic horses than in the tissues from the non-laminitic horses. A comparison of the collagenolytic activity in the laminitic and control tissues showed that collagenolytic activities corresponding to the 92 kDa (P < 0.001), 72 kDa (P < 0.01) and 66 kDa (P < 0.01) bands were induced in the laminitic tissues.     

Neuroleptic use and behavioral disturbance in nursing homes: a 1-year study

This article discusses a longitudinal study of change in disruptive behaviors among nursing home residents treated with neuroleptics compared with those not treated with neuroleptics. Observations were made of 201 participants on admission to and after 1 year in eight skilled nursing facilities. Nine disruptive behaviors were measured using the Psychogeriatric Dependency Rating Scale with nursing assistants. Neuroleptic use was documented from medication records. Odds ratios are reported for the association of behavior at baseline and use of neuroleptics on nine problem behaviors. For those who received neuroleptics during the year, there was greater change in both developing and resolving disruptive behaviors than for those not receiving neuroleptics. For both groups, restless or pacing behavior and belligerent behavior manifested by refusing instructions changed the most, both in developing and in apparently resolving. Our results show that change in disruptive behaviors occurs among nursing home residents regardless of neuroleptic use, but it occurs more frequently among those who receive neuroleptic medication. Knowledge of which disruptive behaviors are most likely to resolve or develop is important in training nursing home staff to cope with the behaviors as well as in planning interventions that may modify such behaviors.     

Alcohol's impact upon glycobiology

This report reviews and illustrates ways in which some of the problems linked to excessive alcohol intake may develop from alcohol-induced alterations of eukaryotic cell surface molecules. Alcohol is the number one drug of abuse in the US, affecting at least 15 million Americans and causing annual losses of more than $80 billion and 100,000 lives. An estimated 20-40% of all persons admitted to general hospitals have alcohol-related problems and are often undiagnosed alcoholics being treated for the consequences of their drinking. Chronic alcohol-related cirrhosis of the liver is the ninth leading cause of death in the US, with over 28,000 deaths annually. Alcohol has harmful effects on almost every organ system in the body, producing cardiovascular disorders, liver disease, neuropathological illness and fetal injury. The etiologic mechanisms for these effects of alcohol is a research area of considerable importance to the National Institute for Alcohol Abuse and Alcoholism.     

Beta 2-adrenergic receptor and angiotensin II receptor modulation of sympathetic neurotransmission in human atria

BACKGROUND: Cultures of epidermal cells are commonly used to study skin biology and differentiation. Recently a method to culture nail matrix cells has been established. OBJECTIVE: We report the biologic characteristics of nail matrix cells in vitro compared with those of epidermal keratinocytes. METHODS: Human nail matrix cells were isolated and cultured in defined medium. Electron-microscopic examination, growth rate, integrin expression and keratin synthesis pattern were evaluated. In addition, the cells were cultured in serum-containing medium. RESULTS: Nail matrix cells appear to be larger than human epidermal keratinocytes and, at the ultrastructural level, they contain a higher euchromatin/heterochromatin ratio and a lower nucleus/cytoplasm ratio and have a higher growth rate. The synthesis of "hard" keratins was detected at all calcium concentrations. Immunofluorescence analyses showed the expression of alpha 2, alpha 3, and alpha 6 integrin subunits. When cultured in serum-containing medium, nail matrix cells produced an outgrowth of epithelium and a spontaneous migration phenomenon associated with a tendency to stratify in a semilunar area that resembles the architecture of the nail matrix. The pluristratified epithelium showed characteristic markers of nail differentiation. CONCLUSION: Culture of nail matrix cells may represent a useful model to study the biologic properties of nail structure, alterations in some nail diseases and the effects of drugs.     

The whistle and the rattle: the design of sound producing muscles

Vertebrate sound producing muscles often operate at frequencies exceeding 100 Hz, making them the fastest vertebrate muscles. Like other vertebrate muscle, these sonic muscles are "synchronous," necessitating that calcium be released and resequestered by the sarcoplasmic reticulum during each contraction cycle. Thus to operate at such high frequencies, vertebrate sonic muscles require extreme adaptations. We have found that to generate the "boatwhistle" mating call (approximately 200 Hz), the swimbladder muscle fibers of toadfish have evolved (i) a large and very fast calcium transient, (ii) a fast crossbridge detachment rate, and (iii) probably a fast kinetic off-rate of Ca2+ from troponin. The fibers of the shaker muscle of rattlesnakes have independently evolved similar traits, permitting tail rattling at approximately 90 Hz.     

Effects of substitutions in the binding surface of an antibody on antigen affinity

The interactions between the Fab and single-chain Fv (scFv) fragments of an antibody (NC10) and its antigen, influenza virus neuraminidase, were analysed in the crystal structures of the Fab-neuraminidase and scFv-neuraminidase complexes. To investigate the contribution to binding made by cavities, salt links and hydrogen bonds in the antibody- antigen interface, 14 single amino acid replacements were made at six contact residues in the scFv fragment by site-directed mutagenesis. The binding affinity of each mutant scFv antibody for neuraminidase was determined with a BIAcore optical biosensor. Four of the mutations resulted in large changes in the free energy of binding to neuraminidase (deltadeltaG > 1 kcal/mol) and together may account for approximately 70% of the free energy of binding. Hence these data support the theory that a small number of residues form the 'functional epitope' and are most important for binding of NC10 to neuraminidase. The salt link between antibody residue (Asp)H56 and (Lys)N432 from neuraminidase was demonstrated to be important for affinity, since substitution of (Asp)H56 with Asn caused a large reduction in the free energy of binding (deltadeltaG = +2.8 kcal/mol). Hydrogen bonds provided by (Tyr)L32 and (Asp)H56 were also important for binding: mutation of (Tyr)L32 to Phe resulted in a significant reduction in binding affinity (deltadeltaG = +1.7 kcal/mol). Disruption of hydrophobic interactions (van der Waals contacts) led to significant reductions in affinity also ((Tyr)H99 to Ala, deltadeltaG = +1.5 kcal/mol; (Leu)L94 to Ala, deltadeltaG > +3.0 kcal/mol). An attempt to increase binding affinity by filling a cavity in the interface with a larger antibody side chain was unsuccessful, as the free energy gained by new antibody-antigen interactions did not compensate for the removal of cavity-bound water molecules.      

Expression of human aldehyde dehydrogenase-3 associated with hepatocellular carcinoma: promoter regions and nuclear protein factors related to the expression

Studies in our laboratory have shown that as early as day 8.5 of development, mouse yolk sac cells can generate T cells when placed in a thymic microenvironment. At this stage, yolk sac cells can also differentiate into myeloid cells in vitro. B cell differentiation in vitro was achieved with day 9 yolk sac by providing a bone marrow stromal feeder layer. We have now established endothelial cell lines and clones from yolk sacs of day 8-12 mouse embryos. These vary in their ability to support stem cell maintenance and differentiation. Our principal work has been carried out with day 12 cloned endothelial cell lines. One clone supported the > 100 fold expansion of yolk sac hematopoietic stem cells that subsequently could generate B cells, T cells and myeloid cells both in vitro and in vivo. Preliminary experiments with endothelial cells from younger embryos are also described.     

Molecular analysis of the third component of canine complement (C3) and identification of the mutation responsible for hereditary canine C3 deficiency

Genetically determined deficiency of the third component of complement (C3) in the dog is characterized by a predisposition to recurrent bacterial infections and to type 1 membranoproliferative glomerulonephritis. The current studies were undertaken to characterize the cDNA for wild-type canine C3 and identify the molecular basis for hereditary canine C3 deficiency. Amplification, cloning, and sequence analysis indicated that canine C3 is highly conserved in comparison with human, mouse, and guinea pig C3. Southern blot analysis failed to show any gross deletions or rearrangements of DNA from C3-deficient animals. Northern blot analysis indicated that the livers of these animals contain markedly reduced quantities of a normal length C3 mRNA. The full-length 5.1-kb canine C3 cDNA was amplified in overlapping PCR fragments. Sequence analysis of these fragments has shown a deletion of a cytosine at position 2136 (codon 712), leading to a frameshift that generates a stop codon 11 amino acids downstream. The deletion has been confirmed in genomic DNA, and its inheritance has been demonstrated by allele-specific oligonucleotide hybridization.     

Hydatid cyst of the interventricular septum in a 3.5-year-old child

An asymptomatic cardiac cyst located in the interventricular septum was diagnosed in a 3.5-year-old child by echocardiographic findings. Surgical ablation was done and histopathologic analysis confirmed a hydatid cyst. The patient was discharged without symptoms.     

Molecular mapping with functional antibodies localizes critical sites on the human IL receptor common gamma (gamma c) chain

The IL receptor common gamma (gamma c) chain is required for the formation of high affinity cytokine receptor complexes for IL-2, IL-4, IL-7, IL-9, and IL-15, and for signals regulating cell survival, growth, and differentiation. Our current understanding of how gamma c chain associates with multiple ligands and receptor subunits is drawn largely from its structural homology to the human growth hormone (hGH) receptor and known structure of the hGH/hGH receptor complex. These receptors share distinct features in their extracellular portions and are believed to function by a mechanism of ligand-induced association of receptor subunits. Here, we report the first directed mutational analysis of the human gamma c chain by alanine scanning conducted across seven regions likely to contain residues required for intermolecular contact. Functionally distinct, neutralizing anti-gamma c mAbs were employed to define critical residues. One particular mAb, CP.B8, unique in its ability to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced proliferation and high affinity cytokine binding of normal T cells as an intact mAb and as a Fab fragment, localized critical residues to four noncontinuous stretches, namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Notably, these residues form a contiguous patch on the gamma c chain surface in a three-dimensional structural model. These results provide functional evidence for the location of contact points on gamma c chain required for its association with multiple ligands.