Control of Pro-Oxidant Activity of Cupric Ions by Entrapment in Unilamellar Lipid Vesicles

As a demonstration of a potential means of delivering and controlling the biochemical and biological activity of metal ions, cupric ions have been trapped in unilamellar phospholipid vesicles. The activity of these cupric ion-containing vesicles as catalysts of the autoxidation of ascorbate and epinephrine has been investigated. A marked increase in autoxidation rate was observed on release of the cupric ion on addition of detergent. When an azobenzene-containing photochromic lipid was incorporated in the bilayer membrane of the vesicles, the release of cupric ions could be initiated by irradiation with ultraviolet light. In the dark, these vesicles remained stable for at least several weeks. Photo-controlled release of liposomally-entrapped species might find application in areas similar to those where 'caged' reagents are presently used.     

Vesicular-arbuscular mycorrhizal fungi induced alteration in poplar root system morphology

Poplar was grown in a soil either inoculated with Scutellispora calospora, Glomus sp E3 or Glomus caledonium or to which a nutrient solution had been added, in order to determine effects on root morphology. Plants were harvested after 115 days. The lengths of individual roots were measured using image analysis and percentage colonisation was determined for different root orders. Colonisation did not affect plant size but induced large changes in root morphology, with lengths of individual secondary and tertiary roots increased in some cases by up to 100%. Root branching was also increased with number of laterals per unit length of colonised roots being up to 6 times greater than in non-colonised roots. These results clearly show that colonisation of roots by Vesicular-arbuscular mycorrhizal fungi can result in significant alteration to poplar root system morphology. They also suggest that the mechanisms of alteration are not entirely due to improved host plant nutrition.     

The construction of human somatic cell hybrids containing portions of the mouse X chromosome and their use to generate DNA probes via interspersed repetitive sequence polymerase chain reaction

Interspersed repetitive sequence polymerase chain reaction (IRS-PCR) has become a powerful tool for the rapid generation of DNA probes from human chromosomes present in rodent somatic cell hybrids. We have constructed a somatic cell hybrid containing a major portion of the mouse X chromosome in a human background (clone 8.0). IRS-PCR was developed for the specific amplification of mouse DNA using either of two primers from the rodent-specific portion of the murine B1 repeat. Amplification was subsequently performed with clone 8.0 and a subclone, 8.1/1, which retains a small murine X-chromosomal fragment including Hprt and the Gdx locus. A total of 15-20 discrete PCR products ranging in size from less than 500 to greater than 3000 bp were obtained from clone 8.0 with each primer. In clone 8.1/1, a subset of these bands plus some additional bands were observed. Nine bands amplified from clone 8.1/1 have been excised from gels and used as probes on Southern blots. All of the fragments behaved as single-copy probes and detected domesticus/spretus variation. They have been regionally mapped using an interspecific backcross. The probe locations are compatible with those of markers known to be present in clone 8.1/1. These results demonstrate the feasibility of this method as applied to the mouse genome and the high likelihood of generating useful DNA probes from a targeted region.     

Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion.

The human lymphocyte homing receptor, LAM-1, mediates the adhesion of lymphocytes to specialized high endothelial venules (HEV) of peripheral lymph nodes. We now report that LAM-1 is also a major mediator of leukocyte attachment to activated human endothelium. In a novel adhesion assay, LAM-1 was shown to mediate approximately 50% of the adhesion of both lymphocytes and neutrophils to TNF-activated human umbilical vein endothelial cells at 4 degrees C. The contribution of LAM-1 to leukocyte adhesion was only detectable when the assays were carried out under rotating (nonstatic) conditions, suggesting that LAM-1 is involved in the initial attachment of leukocytes to endothelium. In this assay at 37 degrees C, essentially all lymphocyte attachment to endothelium was mediated by LAM-1, VLA-4/VCAM-1, and the CD11/CD18 complex, whereas neutrophil attachment was mediated by LAM-1, endothelial-leukocyte adhesion molecule-1, and CD11/CD18. Thus, multiple receptors are necessary to promote optimal leukocyte adhesion to endothelium. LAM-1 also appeared to be involved in optimal neutrophil transendothelial migration using a videomicroscopic in vitro transmigration model system. LAM-1-dependent leukocyte adhesion required the induction and surface expression of a neuraminidase-sensitive molecule that was expressed for at least 24 h on activated endothelium. Expression of the LAM-1 ligand by endothelium was optimally induced by LPS and the proinflammatory cytokines TNF-alpha and IL-1 beta, whereas IFN-gamma and IL-4 induced lower levels of expression. The LAM-1 ligand on HEV and cytokine treated endothelium may be similar carbohydrate-containing molecules, because phosphomannan monoester core complex from yeast Hansenula hostii cell wall blocked binding of lymphocytes to both cell types, and identical epitopes on LAM-1-mediated lymphocyte attachment to HEV and activated endothelium. Thus, LAM-1 and its inducible endothelial ligand constitute a new pair of adhesion molecules that may regulate initial leukocyte/endothelial interactions at sites of inflammation.     

Joint moment and muscle power output characteristics of below knee amputees during running: the influence of energy storing prosthetic feet

Stance phase joint moments, muscle power outputs and mechanical energy characteristics were determined in five normal and five below knee amputee subjects running at 2.8 m s-1. The amputees were studied sequentially on three different prosthetic feet: the SACH foot (solid ankle cushion heel), and two energy storing feet, Seattle and Flex. While wearing the SACH foot, the amputees exhibited major alterations in the distribution and magnitude of muscle power output and muscle work: (1) the total work done by the lower extremity was reduced; (2) the hip extensors became the main source of energy absorption and generation, while in normal subjects the ankle plantarflexors were the major energy generators and the knee extensors the major energy absorbers; (3) the eccentric and concentric knee extensor power outputs were reduced and an abnormal concentric knee flexor power output was noted immediately after heel contact. In four of the amputees, energy storing feet resulted in improvements in the power output and mechanical work characteristics of the lower extremity: (1) the energy storing prosthetic feet generated 2-3 times greater energy than the SACH foot; (2) with the Flex foot the amputees exhibited a more normal pattern and magnitude of hip and knee extensor muscle work. One of the subjects, however, exhibited increased abnormalities with the energy storing prosthetic feet. The amount of energy restored relative to the amount of energy absorbed by each of the prosthetic feet was greater with the energy storing feet than the SACH foot (Flex 84%, Seattle 52%, SACH 31%).     

Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region.

The human transmembrane molecule LAR is a protein tyrosine phosphatase (PTPase) with a cell adhesion molecule-like extracellular receptor region. The structure of LAR hinted at its involvement in the regulation of tyrosine phosphorylation through cell-cell or cell-matrix interactions. We show here that LAR is expressed on the cell surface as a complex of two non-covalently associated subunits derived from a proprotein. The LAR E-subunit contains the cell adhesion molecule-like receptor region, while the LAR P-subunit contains a short segment of the extracellular region, the transmembrane peptide and the cytoplasmic PTPase domains. Proprotein processing occurs intracellularly. Analysis of LAR mutants suggested that cleavage occurs in the LAR extracellular region at a paired basic amino acid site by a subtilisin-like endoprotease. A single amino acid substitution at this site blocked LAR proprotein cleavage. The LAR E-subunit is shed during cell growth, suggesting that LAR receptor shedding may be a mechanism for regulating PTPase function. The use of immunohistochemistry techniques on human tissues demonstrated the expression of LAR by various cell lineages, including epithelial cells, smooth muscle cells and cardiac myocytes. The LAR gene is mapped to chromosome 1, region p32-33, which contains candidate tumor suppressor genes.     

Mapping of X chromosome translocation breakpoints in females with Duchenne muscular dystrophy with respect to exons of the dystrophin gene

There are rare female patients who suffer from Duchenne or Becker muscular dystrophy because they carry an X;autosome translocation with a breakpoint in the dystrophin gene. We have defined the positions of seven of these breakpoints with respect to exon-containing HindIII fragments detected by dystrophin cDNA. One breakpoint lies between exon-containing Hindlll fragments 7 and 8, five breakpoints between exon-containing HindIII fragments 31 to 41, and one lies close to exon-containing-HindIII fragment 50. The distribution of these and of a further seven translocation breakpoints whose positions are known is compared with that reported for deletions and duplications in affected males.     

Sequences within and adjacent to the transmembrane segment of alpha-2,6-sialyltransferase specify Golgi retention.

The glycosyltransferase alpha-2,6-sialyltransferase (ST) is a Type II membrane protein localized to the Golgi apparatus. The first 44 amino acids of this protein were able to specify Golgi retention of a fused marker protein, lysozyme. This section of ST contains a transmembrane segment which serves as a non-cleaved signal anchor. When lysozyme was fused to an equivalent region of a cell surface protein it now appeared on the cell surface. Analysis of chimeras between the two proteins revealed that the transmembrane segment of ST specifies Golgi retention. Furthermore, altering this segment in full-length ST results in the protein accumulating on the cell surface. However, the retaining effect of the transmembrane domain of ST is augmented by the presence of adjacent lumenal and cytoplasmic sequences from ST. If these sequences are spaced apart by a transmembrane domain of the same length as that of ST they too can specify Golgi retention. Thus retention in the Golgi of ST appears to involve recognition of an extended region of the protein within and on both sides of the bilayer.     

Regional localization of human ecto-5′nucleotidase to chromosome 6q14–q21

Summary Human and mouse hybrids that contain fragments of human chromosome 6 as translocations were analysed for expression of ecto-5nucleotidase enzymic activity measured by the conversion of AMP to adenosine and for antigenicity recognized by a monoclonal antibody specific for the human isozyme. Both methods allow a regional assignment of ecto-5nucleotidase to 6q14–q21.     

Sesquiterpenes from the marine red alga Laurencia distichophylla

A new cuparene-type sesquiterpene, isolaurenisol, has been isolated and identified from the New Zealand red alga Laurencia distichophylla. Major differences in the chemical composition of two morphologically indistinguishable samples of L. distichophylla are noted.