Biomedical subjects

K M Johansen

Publications and source records attributed to K M Johansen.

At least 19 recordsLinked to original sources

PubMed2001

Molecular identification and sequence analysis of Hillarin, a novel protein localized at the axon hillock.

The monoclonal antibody Lan3-15 identifies a novel protein, Hillarin, that is localized to the axon hillock of leech neurons. Using this antibody we have identified a full length cDNA coding for leech Hillarin and determined its sequence. The gene encodes a 1274 residue protein with a predicted molecular mass of 144013 Da. Data base searches revealed that leech Hillarin has potential orthologues in fly and nematode and that these proteins share two novel protein domains. The W180 domain is characterized by five conserved tryptophans whereas the H domains share 21 invariant residues. In contrast to the arrangement in fly and nematode the cassette containing the W180 and H domains is repeated twice in leech Hillarin. This suggests that the leech Hillarin sequence originated from a duplication event of an ancestral protein with single cassette structure.

Amino Acid Sequence↗

PubMed2001

The JIL-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila.

To analyze the function of the chromosomal kinase JIL-1, we generated an allelic series of hypomorphic and null mutations. JIL-1 is an essential kinase for viability, and reduced levels of JIL-1 kinase activity lead to a global change in chromatin structure. In JIL-1 hypomorphs, euchromatic regions of polytene chromosomes are severely reduced and the chromosome arms condensed. This is correlated with decreased levels of histone H3 Ser10 phosphorylation. These levels can be restored by a JIL-1 transgene placing JIL-1 directly in the pathway mediating histone H3 phosphorylation. We propose a model where JIL-1 kinase activity is required for maintaining the structure of the more open chromatin regions that facilitate gene transcription.

Alleles↗

PubMed2000

Skeletor, a novel chromosomal protein that redistributes during mitosis provides evidence for the formation of a spindle matrix.

A spindle matrix has been proposed to help organize and stabilize the microtubule spindle during mitosis, though molecular evidence corroborating its existence has been elusive. In Drosophila, we have cloned and characterized a novel nuclear protein, skeletor, that we propose is part of a macromolecular complex forming such a spindle matrix. Skeletor antibody staining shows that skeletor is associated with the chromosomes at interphase, but redistributes into a true fusiform spindle structure at prophase, which precedes microtubule spindle formation. During metaphase, the spindle, defined by skeletor antibody labeling, and the microtubule spindles are coaligned. We find that the skeletor-defined spindle maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate. Consequently, the properties of the skeletor-defined spindle make it an ideal substrate for providing structural support stabilizing microtubules and counterbalancing force production. Furthermore, skeletor metaphase spindles persist in the absence of microtubule spindles, strongly implying that the existence of the skeletor-defined spindle does not require polymerized microtubules. Thus, the identification and characterization of skeletor represents the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus before microtubule spindle formation.

Active Transport, Cell Nucleus↗

PubMed2000

Posttranslational processing and differential glycosylation of Tractin, an Ig-superfamily member involved in regulation of axonal outgrowth.

Tractin is a novel member of the Ig-superfamily which has a highly unusual structure. It contains six Ig domains, four FNIII-like domains, an acidic domain, 12 repeats of a novel proline- and glycine-rich motif with sequence similarity to collagen, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ domain binding motif. By generating domain-specific antibodies, we show that Tractin is proteolytically processed at two cleavage sites, one located in the third FNIII domain, and a second located just proximal to the transmembrane domain resulting in the formation of four fragments. The most NH(2)-terminal fragment which is glycosylated with the Lan3-2, Lan4-2, and Laz2-369 glycoepitopes is secreted, and we present evidence which supports a model in which the remaining fragments combine to form a secreted homodimer as well as a transmembrane heterodimer. The extracellular domain of the dimers is mostly made up of the collagen-like PG/YG-repeat domain but also contains 11/2 FNIII domain and the acidic domain. The collagen-like PG/YG-repeat domain could be selectively digested by collagenase and we show by yeast two-hybrid analysis that the intracellular domain of Tractin can interact with ankyrin. Thus, the transmembrane heterodimer of Tractin constitutes a novel protein domain configuration where sequence that has properties similar to that of extracellular matrix molecules is directly linked to the cytoskeleton through interactions with ankyrin.

Animals↗

PubMed2000

JIL-1, a chromosomal kinase implicated in regulation of chromatin structure, associates with the male specific lethal (MSL) dosage compensation complex.

JIL-1 is a novel chromosomal kinase that is upregulated almost twofold on the male X chromosome in Drosophila. Here we demonstrate that JIL-1 colocalizes and physically interacts with male specific lethal (MSL) dosage compensation complex proteins. Furthermore, ectopic expression of the MSL complex directed by MSL2 in females causes a concomitant upregulation of JIL-1 to the female X that is abolished in msl mutants unable to assemble the complex. Thus, these results strongly indicate JIL-1 associates with the MSL complex and further suggests JIL-1 functions in signal transduction pathways regulating chromatin structure.

Animals↗

PubMed2000

Ectopic CNS projections guide peripheral neuron axons along novel pathways in leech embryos.

Previous studies have indicated that the formation of stereotyped segmental nerves in leech embryos depends on the interactions between CNS projections and ingrowing afferents from peripheral neurons. Especially, CNS-ablation experiments have suggested that CNS-derived guidance cues are required for the correct navigation of several groups of peripheral sensory neurons. In order to directly test this hypothesis we have performed transplantations of CNS ganglia into ectopic sites in segments from which the resident ganglia have been removed. We find that the transplanted ganglia extend numerous axons distributed roughly equally in all directions. When these CNS projections reach and make contact with peripheral sensory axons they are used as guides for peripheral neurons to grow toward and into the ectopic ganglia even when this means following novel pathways that cross the midline and/or segmental boundaries. The peripheral sensory axons turn and grow toward the ectopic ganglia only when in physical contact with CNS axons, suggesting that diffusible chemoattractants are not a factor. These results demonstrate that the guidance cues provided by ectopic CNS projections are both necessary and sufficient to steer peripheral sensory neuron axons into the CNS.

Animals↗

PubMed1999

The effect of red wine on the fibrinolytic system and the cellular activation reactions before and after exercise.

The effect of red wine drinking was tested on fibrinolytic parameters and blood cells in nine healthy students at rest and after acute exercise. The subjects were randomly assigned in a crossover design to one of three treatment regimes: control situation, low-dose wine group, and high-dose wine group. Blood samples were drawn just prior to experimental start, at 2 and 4 hours, and the next morning at 8:00 a.m., at 8:30 a.m. just after exercise, and 2 hours after exercise. The fibrinolytic potential was measured by whole blood clot lysis time (WBCLT), tissue plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1) antigens in plasma. A whole blood system was used to test the reactivity of blood cells by stimulating hirudinized blood with 5 ng/mL lipopolyusaccharide (LPS) for 2 hours at 37 degrees C and measurements of tissue necrosis factor alpha and interleukin-8 (IL-8) in the plasma. Intake of red wine caused impaired fibrinolysis shown by prolonged WBCLT (3.6, 20.7, and 55.7%, respectively, for control, low- and high-dose wine groups) due to increase in PAI-1 antigen (-0.8, 4.8, and 11.0 ng/mL, respectively, in the three groups). There was no effect of the red wine the next morning on the fibrinolytic system. A strong correlation was observed between WBCLT and PAI-1 antigen (p<0.0001). Acute exercise caused an immediate rise in both tissue plasminogen activator antigen and PAI-1 antigen levels and WBCLT was significantly shortened. In contrast to that of the wine groups, 2 hours after exercise WBCLT was prolonged in the control group, but not significantly so. Thus the red wine has a negative effect on the fibrinolytic system during rest, but may have a positive effect after strenuous exercise. The red wine had no immediate effect on LPS-induced tissue necrosis factor alpha or IL-8 production, although there was a tendency for higher cytokine production in the control group compared to the wine groups during and just after intake of wine. The next morning after exercise, the LPS-induced IL-8 production increased 137, 89, and 96%, respectively, in control, low-, and high-dose wine groups, probably due to a rise in epinephrine and activation of platelets. Although not significantly so, there was a tendency for red wine intake in the evening to suppress the reactivity of the cells after physical exercise the subsequent morning. It is suggested that the negative effect of red wine ingestion may be due to the toxic effect of ethanol on hepatocytes or adipose tissue and subsequent release of PAI-1, whereas the positive effect may be due to the red wine suppression of platelet activation and release of PAI-1 from activated platelets. It is proposed that at least part of the beneficial effect of red wine ingestion may be associated with the downregulation of cytokine production.

Adult↗

PubMed1999

Differential glycosylation and proteolytical processing of LeechCAM in central and peripheral leech neurons.

LeechCAM is a recently described member of the Ig-superfamily which has five Ig-domains, two FNIII-domains, a transmembrane domain, and a cytoplasmic domain. Phylogenetic analysis indicated that LeechCAM is the leech homolog of apCAM, FasII, and vertebrate NCAM. Using a leechCAM-specific monoclonal antibody we show by immunoblot analysis and by Triton X-114 phase separation experiments that in addition to existing in a transmembrane version LeechCAM is likely to be proteolytically cleaved into a secreted form without the transmembrane domain and the intracellular tail. Furthermore, by immunoprecipitation we demonstrate that LeechCAM is glycosylated with the Laz2-369 glycoepitope, an epitope that has been specifically implicated in regulation of axonal outgrowth and synapse formation.

Animals↗

PubMed1999

Antibody identification, chromosome map assignment, and sequence analysis of a Rab escort protein homolog in Drosophila1.

Using a polyclonal antiserum a cDNA encoding a Rab escort protein (REP) homolog in Drosophila has been identified and sequenced. The gene encodes a 511 residue protein with a predicted molecular mass of 56855 Da. Antibody labeling demonstrates that Drosophila REP protein is present in the early embryo and that it is being apportioned uniformly throughout the embryo in a process likely to be linked to the syncytial nuclear divisions. In situ hybridization to polytene chromosomes reveals that the Drosophila REP gene is located in the 56E region on the second chromosome. Drosophila REP is the first invertebrate REP homolog to be identified and characterized.

Alkyl and Aryl Transferases↗

PubMed1999

Molecular cloning and characterization of LKv1, a novel voltage-gated potassium channel in leech.

We have cloned a novel voltage-gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage-gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I(K). Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage-gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker-related K channels in jellyfish. Thus, this analysis indicates that this group of voltage-gated K channels contains several evolutionarily divergent lineages.

Amino Acid Sequence↗

PubMed1999

Gliarin and macrolin, two novel intermediate filament proteins specifically expressed in sets and subsets of glial cells in leech central nervous system.

Using monoclonal antibodies, we have identified two novel intermediate filament (IF) proteins, Gliarin and Macrolin, which are specifically expressed in the central nervous system of an invertebrate. The two proteins both contain the coiled-coil rod domain typical of the superfamily of IF proteins flanked by unique N- and C-terminal domains. Gliarin was found in all glial cells including macro- and microglial cells, whereas Macrolin was expressed in only a single pair of giant connective glial cells. The identification of Macrolin and Gliarin together with the characterization of the strictly neuronal IF protein Filarin in leech central nervous system demonstrate that multiple neuron- and glial-specific IFs are not unique to the vertebrate nervous system but are also found in invertebrates. Interestingly, phylogenetic analysis based on maximum parsimony indicated that the presence of neuron- and glial cell-specific IFs in coelomate protostomes as well as in vertebrates is not of monophyletic origin, but rather represents convergent evolution and appears to have arisen independently.

Amino Acid Sequence↗

PubMed1999

JIL-1: a novel chromosomal tandem kinase implicated in transcriptional regulation in Drosophila.

We have cloned and characterized JIL-1, a novel tandem kinase in Drosophila that associates with the chromosomes throughout the cell cycle. Antibody staining and live imaging of JIL-1-GFP transgenic flies show that JIL-1 localizes to the gene-rich interband regions of larval polytene chromosomes and is upregulated almost 2-fold on the hypertranscribed male X chromosome compared to autosomes. Phylogenetic analysis suggests that JIL-1 together with human MSKs defines a separate family of tandem kinases. That JIL-1 is a functional kinase was demonstrated by autophosphorylation and phosphorylation of histone H3 in vitro. Based on these findings, we propose that JIL-1 may play a role in transcriptional control potentially by regulating chromatin structure.

Amino Acid Sequence↗

PubMed1999

Chromatin structure and nuclear remodeling.

Nuclear architecture is remodeled during interphase in response to changes in gene activity as well as to changing structural and functional requirements during cell division. Using the monoclonal antibody mAb2A, we have identified two proteins that appear to play important roles in these processes: JIL-1 is a tandem serine-threonine kinase implicated in the regulation of chromatin structure, whereas Skeletor is a novel protein participating in structural nuclear remodeling during the cell cycle. Antibody staining and live imaging of JIL-1-GFP transgenic flies show that JIL-1 localizes to the gene-rich interband regions of larval polytene chromosomes and is upregulated almost twofold on the hypertranscribed male X chromosome compared with autosomes. We propose that JIL-1 may play a role in transcriptional control potentially by regulating chromatin structure. The other mAb2A antigen, Skeletor, is distributed in a nuclear meshwork pattern that can be observed in stereo pair images to reorganize during the cell cycle to form a spindle-like structure at prometaphase that is distinct from the microtubule spindle apparatus. Taking advantage of the powerful molecular and genetic approaches offered in Drosophila, the study of these two proteins promises to yield new insight into what defines nuclear architecture at the molecular level and how its remodeling is regulated.

Amino Acid Sequence↗

PubMed1998

Development and pathway formation of peripheral neurons during leech embryogenesis.

By labeling the germinal plates of staged leech embryos with monoclonal antibodies to the immunoglobulin superfamily member Tractin, we have documented the distribution and initial development of peripheral neurons in a hirudinid leech. We find, in addition to sensillar and extrasensillar sensory neurons, that there are 21 identifiable peripheral neurons in each hemisegment. These neurons are found in highly stereotyped positions, and all but two of them are associated with the segmental nerves. We show that eight of the peripheral neurons have the characteristic morphology of stretch receptor neurons and that they form a circumferentially distributed grid aligned in such a way that each of five specialized longitudinal muscle fascicles are monitored by at least two stretch receptor cells covering ventral, lateral, and dorsal regions of the body wall. Furthermore, we show that, in contrast to the dorsal posterior nerve, which is pioneered by central projections, the pathways of the three remaining segmental nerves are likely to be pioneered or guided by peripheral neurons.

Animals↗

PubMed1997

Differential glycosylation of tractin and LeechCAM, two novel Ig superfamily members, regulates neurite extension and fascicle formation.

By immunoaffinity purification with the mAb Lan3-2, we have identified two novel Ig superfamily members, Tractin and LeechCAM. LeechCAM is an NCAM/FasII/ApCAM homologue, whereas Tractin is a cleaved protein with several unique features that include a PG/YG repeat domain that may be part of or interact with the extracellular matrix. Tractin and LeechCAM are widely expressed neural proteins that are differentially glycosylated in sets and subsets of peripheral sensory neurons that form specific fascicles in the central nervous system. In vivo antibody perturbation of the Lan3-2 glycoepitope demonstrates that it can selectively regulate extension of neurites and filopodia. Thus, these experiments provide evidence that differential glycosylation can confer functional diversity and specificity to widely expressed neural proteins.

Amino Acid Sequence↗

PubMed1997

Molecular mechanisms mediating axon pathway formation.

During nervous system formation nerve cells extend axons in order to form precise patterns of neuronal connectivity. These connections are often established after the neuronal growth cones have pioneered or navigated through complex pathways to their target area both within the CNS and to and from the periphery. Recent studies have provided evidence that the process of specific pathway formation may rely on a number of molecular guidance mechanisms and cues such as selective adhesion, growth cone avoidance, surface gradients, guidepost cells, and chemotropism. Analysis of the molecular basis for these guidance mechanisms show that the molecules involved often belong to distinct multigene families and that they can provide both short- and long-range attractive as well as repulsive cues. Many of these molecules have a modular structure that is made up of different tandemly arranged domains that allow for multiple functional interactions with a range of other molecules. This allows the same molecule to be multifunctional, for example, by attracting certain neurons while repelling others. This review is an overview of the molecular structure, as it relates to function and mechanisms of action of some of the major gene families thought to be mediating specific axonal guidance and pathway formation.

Animals↗

PubMed1996

Remodeling of nuclear architecture during the cell cycle in Drosophila embryos.

Little is known about what determines the nuclear matrix or how its reorganization is regulated during mitosis. In this study we report on a monoclonal antibody, mAb2A, which identifies a novel nuclear structure in Drosophila embryos which forms a diffuse meshwork at interphase but which undergoes a striking reorganization into a spindle-like structure during pro- and metaphase. Double labelings with alpha-tubulin and mAb2A antibodies demonstrate that the microtubules of the mitotic apparatus co-localize with this mAb2A labeled structure during metaphase, suggesting it may serve a role in microtubule spindle assembly and/or function during nuclear division. That the mAb2A-labeled nuclear structure is essential for cell division and/or maintenance of nuclear integrity was directly demonstrated by microinjection of mAb2A into early syncytial embryos which resulted in a disintegration of nuclear morphology and perturbation of mitosis.

Animals↗

PubMed1996

Initial formation and secondary condensation of nerve pathways in the medicinal leech.

Invertebrates have proved to be important experimental systems for examining questions related to growth cone navigation and nerve formation, in large part because of their simpler nervous systems. However, such apparent simplicity can be deceiving because the final stereotyped patterns may be the result of multiple developmental mechanisms and not necessarily the sole consequence of the pathway choices of individual growth cones. We have examined the normal sequence of events that are involved in the formation of the major peripheral nerves in leech embryos by employing (1) an antibody directed against acetylated tubulin to label neurons growing out from the central nervous system, (2) the Lan3-2 antibody to label a specific population of peripheral neurons growing into the central nervous system, and (3) intracellular dye filling of single cells. We found that the mature pattern of nerves was characterized by a pair of large nerve roots, each of which branched into two major tracts. The earliest axonal projections did not, however, establish this pattern definitively. Rather, each of the four nerves initially formed as discrete, roughly parallel tracts without bifurcation, with the final branching pattern of the nerve roots being generated by a secondary condensation. In addition, we found that some of the nerves were pioneered in different ways and by different groups of neurons. One of the nerves was established by central neurons growing peripherally, another by peripheral neurons growing centrally. These results suggest that the formation of common nerves and neuronal pathfinding in the leech involves multiple sets of growth cone guidance strategies and morphogenetic mechanisms that belie its apparent simplicity.

Afferent Pathways↗