Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

Proteomic changes in Escherichia coli TG1 after metabolic engineering for enhanced trichloroethene biodegradation

Through metabolic engineering, new enzymatic pathways can be introduced into cells to enable or enhance production or biotransformation of chemicals. However, these changes have physiological consequences that can be important but are not well understood. Here we describe the use of two-dimensional gel electrophoresis (2-DE) to detect changes in the proteome of Escherichia coli cells that have been engineered to transform the pollutant trichloroethene (TCE) with the enzyme toluene o-monooxygenase (TOM). Comparison of 2-DE gels (isoelectric point range 4-7) for E. coli cells with and without the ability to synthesize TOM revealed 31 new proteins in TOM-containing cells as well as nine proteins not detected in those cells but present in the plasmid control strain. Exposure of TOM-containing cells to TCE led to the synthesis of four new proteins and the loss of only one protein. Thus, this example of metabolic engineering has a substantial and complex impact on the physiology of these cells that was clearly revealed using a proteomic approach.     

Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking

Huntingtin interacting protein 1 (HIP1) is a recently identified component of clathrin-coated vesicles that plays a role in clathrin-mediated endocytosis. To explore the normal function of HIP1 in vivo, we created mice with targeted mutation in the HIP1 gene (HIP1(-/-)). HIP1(-/-) mice develop a neurological phenotype by 3 months of age manifest with a failure to thrive, tremor and a gait ataxia secondary to a rigid thoracolumbar kyphosis accompanied by decreased assembly of endocytic protein complexes on liposomal membranes. In primary hippocampal neurons, HIP1 colocalizes with GluR1-containing AMPA receptors and becomes concentrated in cell bodies following AMPA stimulation. Moreover, a profound dose-dependent defect in clathrin-mediated internalization of GluR1-containing AMPA receptors was observed in neurons from HIP1(-/-) mice. Together, these data provide strong evidence that HIP1 regulates AMPA receptor trafficking in the central nervous system through its function in clathrin-mediated endocytosis.     

Vegetation history of mid- to western Ireland in the 2nd millennium a.d.; fresh evidence from tephra-dated palynological investigations

The historic Icelandic tephra layers, from Hekla in a.d. 1104 and Öræfajökull in a.d. 1362 that have been found in four peat profiles obtained from lowland and upland mid to western Irish bogs, provide the dating for high-resolution palynological investigations of regional land use over the last thousand years. Marginal agriculture is investigated through the study of an upland blanket peat and a lowland Atlantic blanket peat. At the lowland site, the landscape has been altered, primarily by removal of hazel scrub, while in the uplands, there has been little scrub woodland throughout the last millennium. Pastoral agriculture has a long, unbroken history at both sites, with a short period of arable agriculture, dated to the early 19th century, detected in the uplands. At the two lowland sites, changes in land use associated with medieval monastic and secular activity were similar but not synchronous. The a.d. 1362 tephra in one lowland profile provides high-resolution dating of the palynological evidence for agricultural collapse in the aftermath of the Black Death. The palynological evidence of late medieval woodland clearance is contrasted with the written record. The effects of 19th century population expansion on land use are considered. A synthesis of regional land use in Ireland during the last thousand years is presented.     

Linkage and LOH studies in 19 cylindromatosis families show no evidence of genetic heterogeneity and refine the CYLD locus on chromosome 16q12–q13

Familial cylindromatosis is an autosomal dominant predisposition to multiple neoplasms of the skin appendages. The susceptibility gene has previously been mapped to chromosome 16q12-q13 and has features of a recessive oncogene/tumour suppressor gene. We have now evaluated 19 families with this disease by a combination of genetic linkage analysis and loss of heterozygosity in cylindromas from affected individuals. All 15 informative families show linkage to this locus, providing no evidence for genetic heterogeneity. Recombinant mapping has placed the gene in an interval of approximately 1 Mb. There is no evidence, between families, of haplotype sharing that might be indicative of common founder mutations.     

A Class VI Unconventional Myosin Is Associated with a Homologue of a Microtubule-binding Protein,Cytoplasmic Linker Protein–170, in Neurons and at the Posterior Pole of Drosophila Embryos

Abstract. Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for cellular organization and function. We report here on molecules that may mediate coordination between the actin and microtubule cytoskeletons. We have identified a 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F unconventional myosin. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue identified of cytoplasmic linker protein (CLIP)–170, a protein that links endocytic vesicles to microtubules. We have named this protein D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, leads us to speculate that these two proteins may functionally link the actin and microtubule cytoskeletons.Global organization of the cell and the coordination of its physiology requires interaction between different cytoskeletal systems. During interphase, a typical eukaryotic cell has microtubules emanating from the centrosome located near the nucleus, which extend to the periphery of the cell, presumably interacting with the cortical actin filament meshwork. Microtubules during interphase are thought to be mainly required for the organization of the membrane systems (e.g., vesicular traffic and organelle movement). The actin-rich cortex is important for maintaining cell shape and for cellular movement.There is increasing evidence of coordination between the actin and the microtubule cytoskeletons (Langford, 1995; Koonce, 1996). Data from a number of systems suggests that many cell types use a combination of microtubule and actin filament–based transport in vesicle and organelle trafficking. It is well established that microtubules are required for long distance transport of cellular components. In contrast, the actin cytoskeleton is thought to be required for more local traffic. The best evidence for transport along both cytoskeletal systems is in neurons. Vesicles appear to be transported along actin filaments in mammalian growth cones (Evans and Bridgman, 1995). Furthermore, gelsolin, which promotes depolymerization of actin filaments, has been shown to inhibit fast axonal transport in this system (Brady et al., 1984). In extruded squid axoplasm, Kuznetsov et al. (1992) observed what appeared to be the same vesicle moving along microtubules and then, subsequently, along microfilaments. Inhibitor studies provide evidence that mitochondria can move along both actin filaments and microtubules in neurons in vivo (Morris and Hollenbeck, 1995). These data support the idea that actin filament and microtubule-based transport cooperate to achieve proper organization of cellular components.The same phenomenon may be occurring in other cell types. In yeast, the mutant phenotype of the MYO2 gene, which encodes an unconventional myosin, is suppressed by overexpression of a kinesin-related protein. These two proteins are colocalized in regions of active growth where a polarized arrangement of actin plays an important role (Lillie and Brown, 1992, 1994). Microtubules are not normally required for this growth. Thus, the basis for suppression is not completely understood. However, the phenotypic suppression suggests that perhaps microtubule-based transport can substitute for actin filament–based transport, under some conditions. In polarized epithelial cells, Fath et al. (1994) have isolated a population of vesicles containing both myosin and microtubule motors. They speculate that proper transport of vesicles relies on both microtubule and actin filament–based transport.Previously, it has been shown that a class VI unconventional myosin, the Drosophila 95F unconventional myosin, transports particles along actin filaments during the syncytial blastoderm stage of Drosophila embryonic development (Mermall et al., 1994). 95F myosin activity is required for normal embryonic development (Mermall and Miller, 1995). 95F myosin is also associated with particulate structures in other cells of the embryo later in development where it may also be involved in actin-based transport. To investigate further the transport catalyzed by 95F myosin, we have begun studies to identify proteins associated with 95F myosin that might be cargoes or regulators. In this work, we have identified a protein that coimmunoprecipitates with 95F myosin. Sequence analysis reveals that this protein is the Drosophila homologue of cytoplasmic linker protein (CLIP)¹–170. CLIP-170 is believed to function as a linker between endocytic vesicles and microtubules (Pierre et al., 1992). We have named this associated protein D-CLIP-190. Colocalization of 95F myosin and D-CLIP-190 at the subcellular level at several times in development and in cultured embryonic cells provides support for the in vivo association of these two proteins. The association of a myosin and a homologue of a microtubule-binding protein suggests that these two proteins may act to coordinate the interaction between actin filaments and microtubules.     

Hyaluronectin is produced by oligodendrocytes and Schwann cells in vitro

A hyaluronectin (HN)-like antigen was found in rat O-2A progenitors and oligodendrocytes, as well as in Schwann cells and in their culture medium. The HN-like antigen secreted in culture supernatants had a higher molecular mass than HN extracted from rat brain at acidic pH. In vitro the secreted HN-like antigen was spontaneously and slowly degraded into species whose Mr was close to that of HN found in acidic brain extract. In brain or nerve neutral pH extracts, both HN-like antigen and HN were present. The high Mr of the secreted antigen, the homology in amino acid sequences between HN and N-terminal domain of PG-M/versican, in addition to a positive hybridization between Schwann cell RNAs and a probe obtained with primers derived from HN sequences also found in versican suggested that HN is closely related to the large proteoglycan PG-M/versican. The presence in Schwann cell extract of a HN mRNA whose Mr was compatible with the size expected for HN showed that HN may be directly secreted by cells and not only the consequence of a proteolytic cleavage. The similarity of HN with PG-M (V3) suggested that HN found in vivo could be the result of an alternative splicing of a single gene. We conclude that HN as other members of the PG-M/versican family is a marker of oligodendrocytes and Schwann cells in culture.     

T Lymphocyte activation results in an increased expression of β-1,4-Galactosyltransferase: Phorbol ester induces a similar enhancement in the absence of mitosis

We previously showed that in vitro activated human T lymphocytes expressed increased amounts of -1,6-branched N-linked oligosaccharides (Lemaire S et al. (1994) J Biol Chem 269: 8069-74), which have been proposed to participate in the regulation of the immune process. In the present paper, we compared the activity and expression of -1,4-galactosyltransferase (GalT), one of the glycosyltransferases involved in the biosynthesis of these -1,6-branched N-linked oligosaccharides, before and after in vitro activation of T lymphocytes after a 40 h treatment with a mixture of phorbol 12-myristate 13-acetate and Phaseolus vulgaris lectin. After treatment, the enzymatic activity of the GalT was significantly increased and immunoblot experiments performed with a monoclonal antibody to human GalT showed an increased intensity of the GalT band at 49 kDa, attributable to an enhancement of GalT mRNA level, as shown by Northern blots. However, treatment of the same T-lymphocytes by phorbol ester alone, which is unable to induce mitosis, resulted in a comparable increase of the expression of GalT. Moreover, these phorbol ester-treated T lymphocytes, analysed by flow cytometry exhibited a two-fold increase in the expression of GalT. Finally, confocal fluorescence microscopy performed on all T lymphocytes (treated or not) showed that the flow cytometric signal of GalT originates from intracellular, Golgi-associated antigen only since no surface GalT was detected.