Biomedical subjects

J Yang

Publications and source records attributed to J Yang.

At least 469 records · Page 26Linked to original sources

PubMed2000

Effects of environmental lighting on early semen production and correlated hormonal responses in turkeys.

Recent work at our institution on lighting turkey males for semen production and correlated changes in plasma luteinizing hormone (LH) and testosterone (T) are summarized in this paper. In sexually mature males, both LH and T are secreted in pulses, with a pulse of LH about 10 min prior to a pulse of T. Pulses of LH and T occurred about every 2 h and were equally distributed between the light (L) and dark (D) portions of a 14 h L:10 h D d. The pattern of secretion and overall concentrations of LH and T were not affected by intermittent photoperiod lighting (1 L:2 D, 8 x d) in comparison to continuous photoperiod lighting (14 L:10 D) lighting. Pulses of LH or T were not entrained by L or D with the intermittent or continuous lighting treatment. To study the interaction of age and lighting treatment, males were exposed to one of two lighting treatments: long-day photoperiods (16 L:8 D) d(-1) from 10 to 12 or 29 wk of age (WOA) (Treatment LDLD) or short-day photoperiods (6 L:18 D d(-1) from 10 or 12 to 29 WOA, then long-day photoperiods (Treatment SDLD). Males in the LDLD treatment attained puberty earlier (25 WOA) than those in the SDLD treatment. In the later treatment, most of the males attained puberty after 29 WOA. Both LH and T were low until 18 WOA in the LDLD males, then both increased to adult levels over the next 2 to 3 wk. In the SDLD males, LH and T were lower than in the LDLD males until 48 h after switching to long-day photoperiods, when both were transiently higher before declining to lower adult levels by 35 WOA. Secretory patterns of LH and T were estimated at 13, 23, and 35 WOA, under both lighting treatments. At 13 WOA, LH and T were secreted in pulses, but levels of both hormones were low and not different between lighting treatments, and none of the birds (0/4) in either treatment were producing semen. At 23 WOA, LH and T were secreted in robust pulses, with the LDLD males having higher concentrations of LH and T than the SDLD males. At 23 WOA, most of the males in the LDLD group (3/4) but none in the SDLD group (0/4) were producing semen. At 35 WOA, 6 wk after photostimulation of the SDLD group, all males (4/4) in both groups were producing semen, and LH and T were at adult levels. However, fewer pulses of T were noted for males in the SDLD treatment.

Animals↗

PubMed2000

Circadian rhythm of the preovulatory surge of luteinizing hormone and its relationships to rhythms of body temperature and locomotor activity in turkey hens.

Simultaneous measurements of plasma LH, body temperature, and locomotor activity were made in laying turkey hens and are reported. Blood samples were remotely collected using a jugular cannula system, and body temperature and locomotor activity were remotely monitored using a radiotelemetry system in freely moving laying turkeys. Under a photoschedule of 14L:10D, the period for preovulatory surges of LH was 25.7 +/- 0.4 h while the periods for peak body temperature and onset of sustained locomotor activity were 24.9 +/- 0.4 and 25.7 +/- 0.5 h, respectively. During exposure to constant light, the periods for preovulatory surges of LH, peak body temperature, and onset of sustained locomotor activity increased to 27.9 +/- 0.9, 26.7 +/- 0.7, and 27.4 +/- 0.7 h, respectively. With the 14L:10D photoschedule, initiation of LH surges was restricted to the scotophase, but after 8 days of constant light, initiation of LH surges had dispersed throughout the 24-h subjective day and night. With constant light, the amplitude of the peak body temperature rhythm decreased, while the duration of the locomotor activity rhythm became broadened and, in some birds, disorganized. Peak body temperature and onset of locomotor activity rhythms and LH surges did not coincide, even though peak body temperature, onset of locomotor activity, and LH surges had similar periods. It is concluded that 1) the photoschedule influences the periods of the LH surge, peak body temperature, and onset of locomotor activity; and 2) a specific or direct relationship between the rhythms of LH surge, body temperature, and locomotor activity remains to be determined in laying turkey hens.

Animals↗

PubMed2000

Cocaine- and amphetamine-regulated transcript peptide in the rat epididymis: an immunohistochemical and electrophysiological study.

Cocaine- and amphetamine-regulated transcript (CART) is a novel family of peptides, of which CART peptide fragments 55-102 and 62-102 are reported to be the endogenous, physiologically active peptides. Immunohistochemical studies with an antiserum directed against the CART peptide fragment 55-102 revealed CART-like immunoreactive (CART-LI) nerve fibers in the rat epididymis. The number was highest in the cauda epididymis and became progressively fewer toward the caput epididymis; the vas deferens exhibited an abundance of CART-LI fibers. Injection of the retrograde tracer Fluorogold (Fluorochrome, Inc., Englewood, CO) to the junction between the vas deferens and cauda epididymis labeled a large number of neurons in the major pelvic ganglion, some of which were CART-positive. Double-labeling the ganglion sections with tyrosine hydroxylase (TH) and CART antisera revealed that CART-LI and TH-LI were expressed in two distinct populations of ganglion cells. Some of the TH-LI cells in the ganglia, however, were covered with web-like CART-LI endings. The effects of CART peptide 55-102, referred to herein as CART, on anion secretion in the form of short circuit currents (Isc) were assessed in cultured epithelia. The CART (1 to 5 microM) applied to the basolateral or apical side of the cultured epithelia caused no significant responses on Isc, whereas lys-bradykinin (1 microM) produced a large Isc response in the same preparations. Our results show that CART-LI is present in a population of rat pelvic ganglion cells, which may give rise to CART-LI nerve fibers as observed in the vas deferens and the epididymis. The biological function of CART in the rat epididymis is not known, but it apparently is not involved in ion secretion across the epithelium.

Amphetamine↗

PubMed2000

Foamy gland high-grade prostatic intraepithelial neoplasia.

A 60-year-old man underwent radical prostatectomy for biopsy-proved adenocarcinoma of the prostate. Histologic examination of the entirely embedded prostatectomy specimen revealed extensive ordinary adenocarcinoma, Gleason's grade 3 + 3 = 6, involving both sides of the gland, and extending into extraprostatic soft tissue at the left base. Adjacent to the carcinoma, and separately, extensive high-grade prostatic intraepithelial neoplasia (PIN) was identified, much of which showed bland nuclei and abundant xanthomatous cytoplasm, identical morphologically to that seen in foamy gland prostate carcinoma. However, unlike foamy gland carcinoma, the foamy glands in the current patient were large, showed papillary infolding, and were associated with a discontinuous layer of basal cells, demonstrated by immunostaining for high-molecular weight cytokeratin. No invasive foamy gland carcinoma was identified in the prostatectomy specimen. Immunostains for Ki-67 showed an increased proliferation rate in foamy high-grade PIN glands when compared with adjacent benign glands. Review of additional outside biopsy material revealed foamy gland high-grade PIN on four of seven needle cores, two of which showed no carcinoma. This patient demonstrates a new subtype of high-grade PIN that is difficult to recognize on needle biopsy. It is important to distinguish foamy gland high-grade PIN from its infiltrating counterpart, and it is critical to recognize because of the association of high-grade PIN with prostate carcinoma.

Adenocarcinoma↗

PubMed2000

Nondepolarizing neuromuscular blockers inhibit the serotonin-type 3A receptor expressed in Xenopus oocytes.

UNLABELLED: Molecular cloning and sequence comparison indicates a high degree of structural homology between muscle nicotinic acetylcholine (nACh) and serotonin-type 3 (5-HT(3A)) receptors, both members of the direct ligand-gated family of ion channels. Because of the structural similarities and common evolutionary origin of these receptors, neuromuscular blockers (competitive nACh antagonists) may demonstrate pharmacologic cross talk and exhibit attributes of 5-HT(3A) receptor antagonists. We examined six clinically-used neuromuscular blockers for their ability to antagonize currents flowing through the 5-HT(3A) receptors in voltage clamped Xenopus oocytes. The neuromuscular blockers reversibly inhibited the 5-HT(3A) receptor-gated current in the rank order potency of (IC50 mean +/- SEM): d-tubocurarine (0.046 +/- 0.003 microM), atracurium (0.40 +/- 0.03 microM), mivacurium (15.1 +/- 2.93 microM), vecuronium (16.3 +/- 2.24 microM), and rocuronium (19.5 +/- 2.31 microM). Gallamine was essentially inactive as a 5-HT(3A) receptor antagonist with an extrapolated IC50 of 1170 microM. We demonstrate that drugs classically known as competitive nACh receptor antagonists also block 5-HT(3A) receptors. It is likely that certain neuromuscular blockers share pharmacological properties with 5-HT(3A) receptor antagonists, such as a reduction in postoperative nausea and vomiting. With careful drug selection, pharmacological cross talk could potentially be used to minimize polypharmacy and optimize patient management. IMPLICATIONS: Muscle nicotinic acetylcholine and serotonin-type 3A (5-HT(3A)) receptors are similar. Therefore neuromuscular relaxants may block 5-HT(3A) receptors. Our pharmacological study demonstrates that neuromuscular relaxants, as with ondansetron, are 5-HT(3A) receptor antagonists. It is likely that certain neuromuscular relaxants exhibit ondansetron-like clinical properties, such as reduction in postoperative nausea and vomiting.

Animals↗

PubMed2000

Regulation of proprioceptive memory by subarachnoid regional anesthesia.

BACKGROUND: Patient perception of limb position during regional anesthesia is frequently incorrect. The existing model ascribes this misperception, or phantom sensation, as a reversion to a fixed, slightly flexed, body schema. A model was developed to evaluate the influence of limb position changes on the incidence of incorrect or phantom sensations during regional anesthesia. METHODS: Forty American Society of Anesthesiologists physical status I-III adult patients undergoing genitourinary procedures under subarachnoid anesthesia were assigned to a lidocaine or bupivacaine treatment group and randomly assigned to one of four time groups (1, 4, 7, and 10 min). After blockade, patients were placed supine and blinded to limb positioning manipulations. One leg was flexed and the contralateral leg extended, with leg positions subsequently reversed at the assigned time point. At 10 min, patients were asked to identify the position of each leg. Percentage of incorrect response was analyzed using a logistic regression model with two independent variables: treatment and time. A supplemental study was undertaken to evaluate the observed difference in incorrect perceptions relative to flexed first versus extended limb first sequencing. RESULTS: The inability to perceive a change in limb position under regional anesthesia is dependent on the time after the block that the position change is initiated in relation to the onset characteristics of the local anesthetic. A phantom sensation of an extended leg position clearly exists. The flexed-first limb has a significantly higher incidence of incorrect or phantom perceptions. CONCLUSION: Proprioceptive memory involves a dynamic neuroplastic imprinting process that is influenced by limb or joint position prior to onset of regional anesthesia. This contrasts with previously held beliefs of a purely fixed body schema.

Adult↗

PubMed2000

Estrogen metabolism and risk of breast cancer: a prospective study of the 2:16alpha-hydroxyestrone ratio in premenopausal and postmenopausal women.

Experimental and clinical evidence suggests that 16alpha-hydroxylated estrogen metabolites, biologically strong estrogens, are associated with breast cancer risk, while 2-hydroxylated metabolites, with lower estrogenic activity, are weakly related to this disease. This study analyzes the association of breast cancer risk with estrogen metabolism, expressed as the ratio of 2-hydroxyestrone to 16alpha-hydroxyestrone, in a prospective nested case-control study. Between 1987 and 1992, 10,786 women (ages 35-69 years) were recruited to a prospective study on breast cancer in Italy, the "Hormones and Diet in the Etiology of Breast Cancer" (ORDET) study. Women with a history of cancer and women on hormone therapy were excluded at baseline. At recruitment, overnight urine was collected from all participants and stored at -80 degrees C. After an average of 5.5 years of follow-up, 144 breast cancer cases and four matched controls for each case were identified among the participants of the cohort. Among premenopausal women, a higher ratio of 2-hydroxyestrone to 16alpha-hydroxyestrone at baseline was associated with a reduced risk of breast cancer: women in the highest quintile of the ratio had an adjusted odds ratio (OR) for breast cancer of 0.58 [95% confidence interval (CI) = 0.25-1.34]. The corresponding adjusted OR in postmenopausal women was 1.29 (95% CI = 0.53-3.10). Results of this prospective study support the hypothesis that the estrogen metabolism pathway favoring 2-hydroxylation over 16alpha-hydroxylation is associated with a reduced risk of invasive breast cancer risk in premenopausal women.

Adult↗

PubMed2000

Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome.

OBJECTIVE: Adult respiratory distress syndrome (ARDS) is responsible for a significant portion of the morbidity and mortality during severe acute pancreatitis. Because inflammatory mediators such as tumor necrosis factor (TNF)-alpha and nitric oxide (NO) produced within the lungs have been implicated in sepsis-induced ARDS, we aimed to determine the role of these mediators in pancreatitis-induced ARDS using a model whereby ascites from animals with pancreatitis is transferred to otherwise healthy animals resulting in pulmonary injury. DESIGN: Prospective, randomized, controlled trial. SETTING: Research laboratory at a university medical school. SUBJECTS: Pathogen-free Sprague-Dawley rats weighing 225-250 g. INTERVENTIONS: Sterile, endotoxin- and cytokine-free pancreatic ascites tested for interleukin (IL)-1beta , TNF-alpha, interferon-gamma, and IL-6 was obtained from rats 18 hrs after the induction of severe, acute pancreatitis. Ascites was subsequently administered intravenously (20 mL/kg) to healthy rats. Sham animals were administered intravenous saline. Healthy animals administered intravenous ascites were randomized to receive a single intraperitoneal injection of the p38 mitogen activated kinase inhibitor CNI-1493 (1 mg/kg) or vehicle. MEASUREMENTS: Pulmonary injury was assessed at 24 hrs by histology and leukocyte and protein concentrations via bronchoalveolar lavage. Pulmonary TNF-alpha protein was detected by immunohistochemistry. Serum nitrite, as a measure of NO production, was measured utilizing the Griess reaction. MAIN RESULTS: After the intravenous administration of pancreatic ascites, the number of leukocytes and the protein concentration within the bronchoalveolar fluid were increased and pulmonary histology was worsened consistent with acute lung injury (all p < .001 vs. sham). Each of these variables of pulmonary injury was lessened in animals receiving CNI-1493 and intravenous ascites (p < .05 vs. vehicle). Pulmonary TNF-alpha protein and serum nitrites were decreased with the administration of CNI-1493 (p < .005 vs. vehicle). CONCLUSIONS: A component of pancreatic ascites other than endotoxin, bacteria, or cytokines (IL-1beta, TNF, interferon-gamma, or IL-6) is capable of inducing ARDS in healthy animals. Inhibition of p38 mitogen activated kinase decreases the pulmonary injury through attenuated production of TNF-alpha and NO suggesting a primary role for these mediators in pancreatitis-induced ARDS.

Analysis of Variance↗

PubMed2000

Prolonged but delayed postischemic hypothermia: a long-term outcome study in the rat middle cerebral artery occlusion model.

Delayed but prolonged hypothermia persistently decreases cell death and functional deficits after global cerebral ischemia in rodents. Postischemic hypothermia also reduces infarction after middle cerebral artery occlusion (MCAO) in rat. Because initial neuroprotection is sometimes transient and may not subserve functional recovery, especially on demanding tasks, the authors examined whether postischemic cooling would persistently reduce infarction and forelimb reaching deficits after MCAO. Male spontaneously hypertensive rats were trained to retrieve food pellets in a staircase test that measures independent forelimb reaching ability. Later, rats underwent 90 minutes of normothermic MCAO, through a microclip, or sham operation. In some rats, prolonged cooling (33 degrees C for 24 hours and then 35 degrees C for 24 hours) began 2.5 hours after the onset of ischemia (60 minutes after the start of reperfusion; n = 17 with subsequently 1 death) or sham procedures (n = 4), whereas untreated sham (n = 4) and ischemic (n = 16 with subsequently 1 death) rats maintained normothermia. An indwelling abdominal probe continually measured core temperature, and an automated fan and water spray system was used to produce hypothermia. One month later rats were reassessed in the staircase test over five days and then killed. The contralateral limb impairment in food pellet retrieval was completely prevented by hypothermia (P = 0.0001). Hypothermia reduced an infarct volume of 67.5 mm3 after untreated ischemia to 35.8 mm3 (P < 0.0001). These findings of persistent benefit encourage the clinical assessment of hypothermia.

Animals↗

PubMed2000

Plaster of Paris as an osteoconductive material for interbody vertebral fusion in mature sheep.

STUDY DESIGN: In adult female sheep, histologic and biomechanical criteria were used to determine whether the osteoconductive performance of plaster of paris would promote the incorporation of the tubular titanium mesh implants used for interbody vertebral fusions. OBJECTIVES: To compare the osteogenicity of plaster of paris with that of autogenous iliac crest bone and bone marrow 6 months after they were loaded into tubular titanium mesh cages and implanted as L3-L5 bridges after L4 corpectomies. SUMMARY OF BACKGROUND DATA: One of the aims of surgery for vertebral pathology is to stabilize the spine by interbody fusions. The morbidity associated with the use of iliac crest autograft bone for fusion grafts prompted trials using plaster of paris as an osteoconductive substrate. METHODS: The total volume of bone that invested the L3-L5 mesh cages after 6 months was quantitated by computed tomography scans. All specimens subsequently were cut into fusion mass segments for biomechanical testing in flexion, extension, compression, and torsion, and then embedded in plastic for sectioning and histomorphometry to determine the trabecular bone volume within the titanium mesh. RESULTS: In each experimental model, implants of plaster of paris were the osteoconductive equal of autogenous iliac crest bone/marrow preparations. The volumes of bone formed around and within the titanium mesh were identical, and the tissues were biomechanically indistinguishable. A partial mechanism was determined by modifying the system for midshaft femoral defects. CONCLUSIONS: In the sheep, a tubular titanium mesh packed with plaster of paris forms an osteoconductive conduit to achieve a biomechanically stable interbody lumbar vertebral fusion.

Animals↗

PubMed2000

Antigenic and genetic stability of bovine immunodeficiency virus during long-term persistence in cattle experimentally infected with the BIV(R29) isolate.

Experimental infection of cattle with bovine immunodeficiency virus (BIV) is characterized by persistent, low levels of virus replication in the absence of clinical disease. A virus neutralization (VN) assay was developed to examine the role of VN antibodies in controlling virus replication in cattle experimentally infected with the BIV(R29) isolate of BIV. All animals developed VN antibody, but there was no correlation between VN titres and restriction of virus replication in vivo. BIV infection did not induce high-titred, cross-neutralizing antibody and there was no evidence for antigenic variation through more than 4 years in vivo. Genetic comparisons among the BIV(R29) inoculum virus and viruses isolated from infected animals identified only limited genetic variation during 4 years in vivo. Moreover, there was no evidence that the observed variation was due to selection. Analyses of genetic diversity in the virus stock used for inoculation indicated a fairly homogeneous population. In the absence of high levels of virus replication and overt clinical disease, there appeared to be little selection of virus variants, resulting in antigenic and genetic stability of BIV(R29) during long-term, persistent infection.

Amino Acid Sequence↗

PubMed2000

Quasiperiodicity and transition to chaos

In coupled Lorenz systems, we find that the three-frequency quasiperiodcity exists in the finite parameter range robustly. We also find the period-doubling bifurcation of the torus and the quasiperiodic windows in superchaos. Based on the separation of the dynamics, corresponding explanations are given by making use of the synchronous dynamics.

Journal Article↗

PubMed2000

Synchronization of chaos in coupled systems

The stability of synchronous chaos of coupled oscillators with diffusive and gradient couplings is investigated. The stability boundaries of all transverse modes can be simultaneously drawn by justifying the boundary of a single mode, according to a scaling relation. Therefore, the distribution of stable and unstable regions can be explicitly shown in control parameter space. Bifurcations through different unstable modes, leading to different spatial orders, are analyzed.

Journal Article↗

PubMed2000

In vitro analysis of roles of a disulfide bridge and a calcium binding site in activation of Pseudomonas sp. strain KWI-56 lipase.

The expression of lipase from Pseudomonas sp. strain KWI-56 (recently reclassified as Burkholderia cepacia) had been found to be dependent on an activator gene (act) downstream of its structural gene (lip). In this work, the mature lipase was synthesized in an enzymatically active form with a cell-free Escherichia coli S30 coupled transcription-translation system by expressing a recombinant lipase gene (rlip) encoding the mature lipase in the presence of its purified activator or by coexpression of rlip and act. The in vitro expression systems were used for studying the folding process of the lipase. The addition of dithiothreitol in the expression systems decreased the activity dramatically without affecting the synthesis level of the lipase, whereas the in vitro-synthesized active lipase was relatively stable even in the presence of dithiothreitol. This phenomenon was further investigated by constructing mutant lipase genes only in vitro by PCR without gene cloning. Replacements of cysteine residues (Cys190 and Cys270) forming a sole putative disulfide bond to serine residues decreased the lipase activity greatly, suggesting that the disulfide bond was essential for the proper folding of the lipase. In addition, replacing Asp242 and Asp288, which were deduced to be part of a Ca(2+) binding site, also greatly decreased the activities of the in vitro-synthesized lipases. The role of the Ca(2+) binding site in the activation of the lipase is also discussed.

Base Sequence↗

PubMed2000

Characterization of the zinc binding activity of the rubella virus nonstructural protease.

The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products. A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X. Liu, S. L. Ropp, R. J. Jackson, and T. K. Frey, J. Virol. 72:4463-4466, 1998). To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF. When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn. Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn(65) binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding. All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV-CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein. Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril. In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.

Amino Acid Sequence↗

PubMed2000

Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by rac1 and phosphatidylinositol-3 kinase activation.

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that causes no currently known pathology in humans. Despite the fact that this virus is of increasing interest to molecular medicine as a vector for gene delivery, relatively little is known about the cellular mechanisms controlling infection. In this study, we have examined endocytic and intracellular trafficking of AAV-2 using fluorescent (Cy3)-conjugated viral particles and molecular techniques. Our results demonstrate that internalization of heparan sulfate proteoglycan-bound AAV-2 requires alphaVbeta5 integrin and activation of the small GTP-binding protein Rac1. Following endocytosis, activation of a phosphatidylinositol-3 (PI3) kinase pathway was necessary to initiate intracellular movement of AAV-2 to the nucleus via both microfilaments and microtubules. Inhibition of Rac1 using a dominant N17Rac1 mutant led to a decrease in AAV-2-mediated PI3 kinase activation, indicating that Rac1 may act proximal to PI3 kinase during AAV-2 infection. In summary, our results indicate that alphaVbeta5 integrin-mediated endocytosis of AAV-2 occurs through a Rac1 and PI3 kinase activation cascade, which directs viral movement along the cytoskeletal network to the nucleus.

Biological Transport↗

PubMed2000

Mutational definition of functional domains within the Rev homolog encoded by human endogenous retrovirus K.

Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a cis-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the approximately 25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.

Amino Acid Sequence↗

PubMed2000

Synergistic interaction of MEK kinase 2, c-Jun N-terminal kinase (JNK) kinase 2, and JNK1 results in efficient and specific JNK1 activation.

Mitogen-activated protein kinases (MAPKs) are activated through cascades or modules consisting of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Investigating the molecular basis of activation of the c-Jun N-terminal kinase (JNK) subgroup of MAPK by the MAPKKK MEKK2, we found that strong and specific JNK1 activation by MEKK2 was mediated by the MAPKK JNK kinase 2 (JNKK2) rather than by JNKK1 through formation of a tripartite complex consisting of MEKK2, JNKK2, and JNK1. No scaffold protein was required for the MEKK2-JNKK2-JNK1 tripartite-complex formation. Expression of JNK1, JNKK2, and MEKK2 significantly augmented the coprecipitation of, respectively, MEKK2-JNKK2, MEKK2-JNK1, and JNKK2-JNK1, indicating that the interaction of MEKK2, JNKK2, and JNK1 is synergistic. Finally, the JNK1 was activated more efficiently in the MEKK2-JNKK2-JNK1 complex than was the JNK1 excluded from the complex. Thus, formation of a signaling complex through synergistic interaction of a MAPKKK, a MAPKK, and a MAPK molecule like MEKK2-JNKK2-JNK1 is likely to be responsible for the efficient, specific flow of information via MAPK cascades.

Animals↗