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Biomedical subjects

R H Lee

Publications and source records attributed to R H Lee.

At least 37 records · Page 2Linked to original sources
PubMed1999

Apoptosis induced by inhibition of contact with extracellular matrix in mouse collecting duct cells.

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation and aspects of cell growth control. Detachment from the matrix epithelial cells induces programmed cell death, and this cell detachment induced apoptosis has been referred to as 'anoikis'. This study was undertaken to determine whether apoptosis is induced by inhibition of contact with extracellular matrix (ECM) in collecting duct cells and to investigate the signaling mechanisms of the process. Upon detachment from ECM, mouse inner medullary collecting duct cells (mIMCD-3) and mouse outer cortical collecting duct cells (M-1), which were derived from an SV40 transgenic mouse, entered into programmed cell death. Forced suspension of mIMCD-3 or M-1 cells did not affect the expression of Bcl-2-related proteins and did not activate c-Jun NH(2)-terminal kinase. Detachment of cells from ECM activated p38 mitogen-activated protein kinase (p38), but its inhibition with SB203580 did not protect cells from anoikis. Detachment of cells from matrix inhibited NF-kappaB activity, and the inhibition of NF-kappaB activity by overexpression of nonphosphorylatable I-kappaB increased detachment-induced apoptotic cell death in M-1 cells. Forced suspension of M-1 cells still activated p53 activity. Caspase-8 was activated during anoikis, but the time course of its activation was in accordance with DNA fragmentation. These results indicate that detachment from ECM induces apoptosis in the kidney collecting duct cells. Changes in expression levels of Bcl-2-related proteins or activation of JNK/p38 kinase are not critical for anoikis. Decrease in NF-kappaB activity and activation of p53 induced by inhibition of interaction with ECM play roles in anoikis in SV-40-transformed collecting duct cells. Caspase-8 is activated during detachment-induced apoptosis, the mechanisms of which are independent of activation of cell death receptors.

Animals↗
PubMed1998

Snf1 kinase connects nutritional pathways controlling meiosis in Saccharomyces cerevisiae.

Glucose inhibits meiosis in Saccharomyces cerevisiae at three different steps (IME1 transcription, IME2 transcription, and entry into late stages of meiosis). Because many of the regulatory effects of glucose in yeast are mediated through the inhibition of Snf1 kinase, a component of the glucose repression pathway, we determined the role of SNF1 in regulating meiosis. Deleting SNF1 repressed meiosis at the same three steps that were inhibited by glucose, suggesting that glucose blocks meiosis by inhibiting Snf1. For example, the snf1Delta mutant completely failed to induce IME1 transcripts in sporulation medium. Furthermore, even when this block was bypassed by expression of IME1 from a multicopy plasmid, IME2 transcription and meiotic initiation occurred at only 10 to 20% of the levels seen in wild-type cells. The addition of glucose did not further inhibit IME2 transcription, suggesting that Snf1 is the primary mediator of glucose controls on IME2 expression. Finally, in snf1Delta cells in which both blocks on meiotic initiation were bypassed, early stages of meiosis (DNA replication and commitment to recombination) occurred, but later stages (chromosome segregation and spore formation) did not, suggesting that Snf1 controls later stages of meiosis independently from the two controls on meiotic initiation. Because Snf1 is known to activate the expression of genes required for acetate metabolism, it may also serve to connect glucose and acetate controls on meiotic differentiation.

Cell Cycle Proteins↗
PubMed1998

Bistability in spinal motoneurons in vivo: systematic variations in rhythmic firing patterns.

In the presence of the monoamines serotonin and norepinephrine, spinal motoneurons can exhibit bistable behavior, in which a brief period of excitatory input evokes prolonged self-sustained firing. A brief inhibitory input returns the cell to the quiescent state. To determine whether motoneurons differ in their capacity for bistable behavior, intracellular recordings were obtained in the decerebrate cat preparation. To enhance the likelihood of encountering bistable behavior, the noradrenergic alpha1 agonist methoxamine was applied to the ventral surface of the cord. The capacity of the cells to produce bistable behavior was assessed from the duration of self-sustained firing evoked by a brief (1.5 s) excitatory synaptic input from muscle spindle Ia afferents. About 35% (17 of 49) of the cells produced steady self-sustained firing for >3 s and were considered fully bistable. The other 32 cells ( approximately 65%) were partially bistable, with self-sustained firing lasting <3 s. Fully bistable cells tended to have lower current thresholds for spike initiation and slower axonal conduction velocities than did partially bistable cells. This suggests that fully bistable motoneurons innervate fatigue resistant muscle fibers. The frequency-current (F-I) relations of the motoneurons were characterized with slow triangular current ramps. Fully bistable cells displayed an acceleration in firing rate immediately on initiation of rhythmic firing. The F-I gain after completion of the acceleration was positive. Fully bistable cells also displayed a hysteresis in the current level for firing threshold with the ascending threshold occurring at substantially higher current level than the descending one. Additionally, these current thresholds usually were centered about zero current, so that the ascending current threshold was positive while the descending current threshold was negative. This negative offset meant that fully bistable cells could exhibit tonic firing without depolarizing injected current. Partially bistable cells exhibited very different F-I characteristics. Firing rate acceleration was just as large as in fully bistable cells but did not occur until well above the current level needed to initiate rhythmic firing. F-I gain after acceleration was negative, there was little to no hysteresis between the ascending and descending firing thresholds, and both thresholds were above the zero current level. These properties of partially bistable cells suggest their functional role is in tasks requiring relatively brief, high forces. The low thresholds of fully bistable cells mean they will be readily recruited in low force tasks like posture, where their prolonged self-sustained firing would be advantageous.

Action Potentials↗
PubMed1998

Bistability in spinal motoneurons in vivo: systematic variations in persistent inward currents.

Bistable behavior in spinal motoneurons consists of self-sustained firing evoked by a brief period of input. However, not all motoneurons possess an equal capacity for bistable behavior. In the companion paper, we found that self-sustained firing was persistent for long periods only in motoneurons with low rheobases and slow axonal conduction velocities. High rheobase, fast conduction velocity motoneurons tend to be only partially bistable in that self-sustained firing lasts at most 1-2 s. The mechanisms underlying these differences between fully and partially bistable motoneurons were investigated by measuring their current voltage (I-V) relationships in the decerebrate cat preparation after administration of the noradrenergic alpha1 agonist methoxamine. Slow (8 mV/s) triangular voltage commands were applied using the discontinuous single-electrode voltage-clamp technique. Both fully and partially bistable cells exhibited a region of negative I-V slope due to activation of a strong, persistent inward current. The peak amplitude of the total persistent inward current (IPIC) was equally large in fully and partially bistable cells, but there were substantial differences in how IPIC was activated and deactivated. In fully bistable cells, the offset of IPIC on the descending phase of the triangular voltage command occurred at a substantially more hyperpolarized voltage then its onset on the rising phase. Thus the I-V function of fully bistable cells exhibited marked hysteresis. Partially bistable cells had significantly less hysteresis. The lack of hysteresis in partially bistable cells was due to a greater decay of IPIC with time than that seen in fully bistable cells. Furthermore, the range over which activation and deactivation of IPIC occurred was more depolarized in partially than in fully bistable cells. The I-V functions were compared with frequency-current (F-I) functions from the same cells, the characteristics of which were presented in the companion paper. The strong onset-offset difference in IPIC in fully bistable cells corresponded to a similarly large hysteresis for the thresholds of their F-I functions. The reduced onset-offset difference for IPIC in partially bistable cells corresponded to a lack of hysteresis in F-I thresholds. Thus the properties of IPIC accounted for the main differences in the F-I behavior seen between fully and partially bistable cells.

Action Potentials↗
PubMed1997

Gene structure and chromosome localization of the G gamma c subunit of human cone G-protein (GNGT2).

Phototransduction in the vertebrate rod and cone photoreceptors is regulated by structurally homologous and yet distinct groups of signaling proteins. We have previously identified in bovine retinas a cone-specific G-protein gamma subunit (G gamma c, previously named G gamma b), which may play a key role in coupling the cone visual pigment to phosphodiesterase (O. C. Ong et al., 1995, J. Biol. Chem. 270:8495-8500). We report here the characterization of human G gamma c and its gene structure. Human G gamma c subunit shares a high degree of sequence identity with the corresponding bovine G gamma c isoform (85%) and human rod G gamma 1 (63%). The protein is specifically localized in cones, as indicated by immunohistochemical staining using anti-G gamma c antibodies. Nucleotide sequence analysis of the G gamma c gene (GNGT2) reveals a structure consisting of three exons and two introns, with the intron splice sites similar to that of the rod G gamma 1 gene (GNGT1). By using fluorescence in situ hybridization, we have further localized the human GNGT2 gene to chromosome 17q21. The elucidation of the G gamma c gene structure would facilitate the identification of genetic defects associated with cone degeneration.

Amino Acid Sequence↗
PubMed1997

Phosducin and betagamma-transducin interaction I: effects of post-translational modifications.

The interaction between phosducin and betagamma-transducin plays regulatory roles in light adaptation of photoreceptors. Both phosducin and betagamma-transducin undergo post-translational modifications, with phosducin modified by phosphorylation and the gamma subunit of betagamma-transducin by farnesylation and carboxylmethylation. In this study we exploited the electrophoretic mobilities of these native proteins to develop a micro binding assay and examined the effects of post-translational modifications on binding affinities. It was found that decarboxylmethylation of gamma-transducin increased the mobility of betagamma-transducin during native gel electrophoresis, but decreased the apparent affinity for phosducin by about 2-fold. Phosphorylation of phosducin by protein kinase A increased the mobility but decreased the apparent affinity for betagamma-transducin by at least 3-fold.

Animals↗
PubMed1997

Patterns of facial laceration from blunt trauma.

The clinical characteristics of craniofacial injuries resulting from various modes of blunt trauma, including motor vehicle accidents, falls, and assault, have been described extensively in the literature. In this study, specifically targeted blunt trauma to selected areas of the face was used to recreate soft-tissue laceration injuries on 19 cadaver heads. The patterns of laceration produced were then examined by location, size, penetrated skin depth, and associated muscle and bony involvement. Results showed reproducible patterns of lacerations on the forehead, bilateral superior orbital rim, nose, perimaxillary region, and chin. Six of 19 cadaver faces were undermined prior to blunt trauma to determine the effects of subcutaneous attachments on laceration patterns. Results showed no consistent difference in laceration patterns between undermined skin and intact skin. Our findings suggest that in response to blunt trauma, the skin breaks along selected lines of least resistance that closely parallel cleavage lines of the face and that the patterns of laceration generated are associated with inherent structural and biomechanical properties of the skin, independent of subcutaneous attachments. Blunt trauma lacerations to the face therefore may occur in a consistent and reproducible manner and may be subject to classification.

Biomechanical Phenomena↗
PubMed1996

Nutritional regulation of late meiotic events in Saccharomyces cerevisiae through a pathway distinct from initiation.

The IME1 gene is essential for initiation of meiosis in the yeast Saccharomyces cerevisiae, although it is not required for growth. Here we report that in stationary-phase cultures containing low concentration of glucose, cells overexpressing IME1 undergo the early meiotic events, including DNA replication, commitment to recombination, and synaptonemal complex formation and dissolution. In contrast, later meiotic events, such as chromosome segregation, commitment to meiosis, and spore formation, do not occur. Thus, nutrients can repress the late stages of meiosis independently of their block of initiation. Cells arrested at this midpoint in meiosis are relatively stable and can resume meiotic differentiation if transferred to sporulation conditions. Resumption of meiosis does not require repression of IME1 expression, since IME1 RNA levels stay high after transfer of the arrested cells to sporulation medium. These results suggest that meiosis in S. cerevisiae is a paradigm of a differentiation pathway regulated by signal transduction at both early and late stages.

Amino Acids↗
PubMed1996

Restoration of extensor excitability in the acute spinal cat by the 5-HT2 agonist DOI.

1. The decerebrate cat preparation with an intact spinal cord is characterized by a high degree of excitability in extensor motoneuron pools, which is eliminated by acute spinalization. Subtype-specific agonists for serotonin (5-HT) were investigated in terms of their effectiveness in restoring the extensor excitability following spinalization. 2. Our hypothesis was that 5-HT2 receptors have the primary role in enhancement of extensor reflex excitability, whereas 5-HT1A and 5-HT1B/D receptors are relatively unimportant. Reflex excitability was assessed from the tonic levels of force and electromyographic (EMG) output from the ankle extensors medial gastrocnemius (MG) and soleus (SOL), and from the reflex forces in both these muscles generated by ramp-and-hold stretches of MG. 3. Before spinal transection, MG and SOL usually exhibited a small amount of tonic background EMG activity and force output. Ramp-and-hold stretch of MG generated a large-amplitude reflex response. Spinal transection at the level of T10 virtually abolished tonic background activity in both extensors and greatly attenuated the MG stretch reflex. Ventral topical application of the selective 5-HT2A/2C agonist (+-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane hydrochloride (DOI) restored the amplitude of the MG stretch reflex in a dose-dependent fashion. However, a considerable portion of the DOI-mediated restoration of MG stretch reflex force was due to elevation of tonic background force levels above previous intact cord levels. 4. The DOI-induced increase in extensor tonic background excitability and facilitation of MG stretch reflex were reversed by ventral topical administration of the selective 5-HT2 antagonist ketanserin. No increase in extensor excitability was observed in spinalized preparations after administration of either the 5-HT1A agonist (+-)-8-hydroxy-dipropylaminotetralin hydrobromide or the 5-HT1B/1D agonist 7-trifluoromethyl-4-(4 methyl-1-piperazinyl)-pyrrolo[1,2- a]quinoxaline maleate. These data strongly suggest that the DOI-induced facilitation of extensor stretch reflex and tonic activity in spinalized preparations is mediated through an action on spinal 5-HT2 receptors. 5. One important difference between the actions of DOI in spinalized versus intact states was that the DOI-induced tonic and reflex forces in the spinalized state were subject to irregular oscillations. In contrast, DOI did not noticeably affect the smoothness of reflex force generation in the intact state. This discrepancy was probably due to the effects of clasp knife inhibition from muscular free nerve endings, which have potent reflex actions in the spinalized but not intact states. Thus DOI elevated excitability levels but did not alter the effects of spinalization on stretch reflex patterns.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1996

Influence of voltage-sensitive dendritic conductances on bistable firing and effective synaptic current in cat spinal motoneurons in vivo.

1. After application of the noradrenergic alpha 1 agonist methoxamine, muscle spindle Ia input evoked bistable firing patterns (i.e., persistent discharges after Ia input ceased) in adult spinal motoneurons in the decerebrate cat preparation. These bistable discharge patterns were compared with the Ia currents generated in voltage-clamp conditions. 2. During voltage clamp at depolarized holding potentials, the Ia effective synaptic current underwent strong amplification. In those cells with strong bistable firing, a prolonged tail current followed the Ia input. Because the voltage clamp held the behavior of somatic conductances constant, these data indicate that voltage-sensitive conductances in the motoneuron dendrites made an important contribution to bistable firing.

Action Potentials↗
PubMed1995

Adenylyl cyclase integrates multiple G protein signals to modulate calcium currents in neonatal rabbit heart.

We investigated the effects of added beta gamma subunits of G proteins (G beta gamma) on beta-adrenergic responsiveness of transmembrane Ca2+ currents (ICa) in ventricular myocytes from neonatal rabbits. G beta 1 gamma 1 purified from retinal rods was dialyzed into cells via the voltage clamp micro-electrode. Stimulation of ICa by isoproterenol was not affected by added intracellular G beta 1 gamma 1 or by carbachol alone but was completely blocked by combined G beta 1 gamma 1 and carbachol. Pretreatment of cells with pertussis toxin or temporal separation of carbachol and isoproterenol allowed stimulation of ICa by isoproterenol in cells dialyzed with G beta 1 gamma 1. Carbachol and G beta 1 gamma 1 together also did not prevent stimulation of ICa by dibutyryl-cyclic AMP. Thus, rather than simply inactivating Gs alpha by mass action, G beta 1 gamma 1 acts in concert with carbachol to inhibit isoproterenol stimulation of ICa.

Adenylate Cyclase Toxin↗
PubMed1995

Molecular cloning and characterization of the G protein gamma subunit of cone photoreceptors.

The phototransduction process in cones has been proposed to involve a G protein that couples the signal from light-activated visual pigment to the effector cyclic GMP phosphodiesterase. Previously, we have identified and purified a G beta gamma complex composed of a G beta 3 isoform and an immunochemically distinct G gamma subunit (G gamma 8) from bovine retinal cones (Fung, B. K.-K., Lieberman, B. S., and Lee, R. H. (1992) J. Biol. Chem. 267, 24782-24788; Lee, R. H., Lieberman, B.S., Yamane, H. K., Bok, D., and Fung, B. K.-K. (1992a) J. Biol. Chem. 267, 24776-24781). Based on the partial amino acid sequence of this cone G gamma 8, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding G gamma 8. The cDNA insert of this clone includes an open reading frame of 207 bases encoding a 69-amino acid protein. The predicted protein sequence of G gamma 8 shares a high degree of sequence identity (68%) with the G gamma (G gamma 1) subunit of rod transducin. Similar to rod G gamma 1, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for G gamma 8 is present at a high level in the retina and at a very low level in the lung, but is undetectable in other tissues. Immunostaining of bovine retinal sections with an antipeptide antibody against the N-terminal region of G gamma 8 further shows a differential localization of G gamma 8 to cones with a pattern indistinguishable from that of G beta 3. This finding suggests that G beta 3 gamma 8 is a component of cone transducin involved in cone phototransduction and color vision.

Amino Acid Sequence↗
PubMed1994

The marine natural product, halistanol trisulfate, inhibits pp60v-src protein tyrosine kinase activity.

Halistanol trisulfate, a sulfated steroid derivative, was isolated from the extracts of two different marine sponges (genus Topsentia) by bioassay-guided fractionation. It exhibited an IC50 of approximately 4 microM against pp60v-src, the oncogenic protein tyrosine kinase encoded by Rous sarcoma virus. Removing the sulfate groups by acid hydrolysis produced the inactive tris-alcohol, halistanol. The kinetics of inhibition were examined and revealed that halistanol trisulfate is a competitive inhibitor with respect to the peptide substrate, [val5]-angiotensin II, and a mixed inhibitor with respect to ATP. A number of monosulfated steroids were studied for protein tyrosine kinase inhibitory activity, but were found to be inactive.

Animals↗
PubMed1994

Muscarinic K+ channels are activated by beta gamma subunits and inhibited by the GDP-bound form of alpha subunit of transducin.

It was reported that transducin beta gamma subunits (T beta gamma) inhibited cardiac muscarinic K+ (KACh) channels activated through GK. This result has been used to support the proposal that GK activates this channel through its alpha subunits. Here, we have reexamined the effects of transducin subunits on KACh channels. T beta gamma applied to the internal side of the inside-out patch membrane activated KACh channels in a concentration-dependent manner (EC50 approximately 1 microM). In contrast, the GDP-bound form of transducin alpha subunits (50 nM-3 microM), but not T beta gamma (1 nM-1 microM), irreversibly inhibited KACh channel openings induced either by GTP plus acetylcholine or by GTP gamma S (guanosine 5'-3-O-(thio)triphosphate). This inhibition was antagonized by exogenously applied T beta gamma (600 nM-3 microM) or brain G protein beta gamma subunits (10-30 nM). These data, opposite to the previous report, indicate that GK activates KACh channels through its beta gamma subunits.

Animals↗
PubMed1994

Structure of malhamensilipin A, an inhibitor of protein tyrosine kinase, from the cultured chrysophyte Poterioochromonas malhamensis.

A new chlorosulfolipid, malhamensilpin A [1] was isolated from the cultured chrysophyte Poterioochromonas malhamensis. Malhamensilipin A was demonstrated to be a modest inhibitor of pp60v-src protein tyrosine kinase. The structure was determined by detailed spectral analysis to be a novel C24 hexachloro lipid containing a vinyl sulfate ester (2,11,12,13,15,16-hexachloro-14-hydroxy-n-tetracos-1E-enol-1-sulfa te).

Bacteria↗