The purpose of this study was to investigate whether the excision repair cross-complementation group 1 (ERCC1) mRNA expression could predict treatment response of patients with locally advanced cervical squamous cell carcinoma (LACSCC) who underwent cisplatin-based concurrent chemoradiotherapy (CCCRT).
A total of sixty LACSCC patients, treated with radical CCCRT from a single institution were evaluated. ERCC1 mRNA expression was determined by quantitative real-time RT-PCR in pre-treatment tumor tissues. The association of ERCC1 status with clinicopathological characteristics (age, histological grade, tumor size, parametrial invasion, lymph node metastasis and FIGO stage) and treatment response were analyzed.
No significant association between ERCC1 mRNA expression and clinicopathological characteristics were observed. Patients with low ERCC1 mRNA level had a significantly higher rate of complete response (86.21%) than patients with high level of ERCC1 expression (19.36%; p < 0.001). In the logistic regression analysis, low ERCC1 mRNA level retained an independent role in predicting complete response to CCCRT (P < 0.001). An ERCC1 expression level of 0.0901 was determined as an optimal cutoff value to identify complete response patients to CCCRT treatment. The sensitivity for detection of a complete response was 81.48% with a specificity of 96.97% (area under the curve, 0.893; 95% confidence interval, 0.804–0.983).
This is the first analysis of the association between ERCC1 mRNA levels and treatment response in patients with LACSCC. Low ERCC1 mRNA level appears to be a highly specific predictor of response to CCCRT in LACSCC.
Excision repair cross-complementation group 1 (ERCC1); Cervical squamous cell carcinoma; Chemoradiotherapy; Response prediction
Many patients with influenza have more than one viral agent with co-infection frequencies reported as high as 20%. The impact of respiratory virus copathogens on influenza disease is unclear. We sought to determine if respiratory virus co-infection with pandemic H1N1 altered clinical disease.
Respiratory samples from 229 and 267 patients identified with and without H1N1 influenza respectively were screened for the presence of 13 seasonal respiratory viruses by multiplex RT-PCR. Disease severity between coinfected and monoinfected H1N1 patients were quantified using a standardized clinical severity scale. Influenza viral load was calculated by quantitative RT-PCR.
Thirty (13.1%) influenza samples screened positive for the presence of 31 viral copathogens. The most prominent copathogens included rhinovirus (61.3%), and coronaviruses (16.1%). Median clinical severity of both monoinfected and co-infected groups were 1. Patients coinfected with rhinovirus tended to have lower clinical severity (median 0), whereas non rhinovirus co-infections had substantially higher clinical severity (median 2). No difference in H1N1 viral load was observed between co-infected and mono infected groups.
Respiratory viruses co-infect patients with influenza disease. Patients coinfected with rhinovirus had less severe disease while non-rhinovirus co-infections were associated with substantially higher severity without changes in influenza viral titer.
Influenza; Influenza Co-infection; Co-infection; Dual Infection; Respiratory Virus Co-infection; Viral Co-infection; Pneumonia; Respiratory Disease
microRNAs (miRNAs) are shown to be involved in the regulation of circadian clock. However, it remains largely unknown whether miRNAs can regulate the core clock genes (Clock and Bmal1).
In this study, we found that mir-142-3p directly targeted the 3’UTR of human BMAL1 and mouse Bmal1. The over-expression (in 293ET and NIH3T3 cells) and knockdown (in U87MG cells) of mir-142-3p reduced and up-regulated the Bmal1/BMAL1 mRNA and protein levels, respectively. Moreover, the expression level of mir-142-3p oscillated in serum-shocked NIH3T3 cells and the results of ChIP and luciferase reporter assays suggested that the expression of mir-142-3p was directly controlled by CLOCK/BMAL1 heterodimers in NIH3T3 cells.
Our study demonstrates that mir-142-3p can directly target the 3’UTR of Bmal1. In addition, the expression of mir-142-3p is controlled by CLOCK/BMAL1 heterodimers, suggesting a potential negative feedback loop consisting of the miRNAs and the core clock genes. These findings open new perspective for studying the molecular mechanism of circadian clock.
mir-142-3p; Bmal1; Circadian clock
The sympathetic nervous system has been implicated in pain associated with painful diabetic neuropathy. However, therapeutic intervention targeted at the sympathetic nervous system has not been established. We thus tested the hypothesis that sympathetic nerve blocks significantly reduce pain in a patient with painful diabetic neuropathy who has failed multiple pharmacological treatments. The diagnosis of small fiber sensory neuropathy was based on clinical presentations and confirmed by skin biopsies. A series of 9 lumbar sympathetic blocks over a 26-month period provided sustained pain relief in his legs. Additional thoracic paravertebral blocks further provided control of the pain in the trunk which can occasionally be seen in severe diabetic neuropathy cases, consequent to extensive involvement of the intercostal nerves. These blocks provided sustained and significant pain relief and improvement of quality of life over a period of more than two years. We thus provided the first clinical evidence supporting the notion that sympathetic nervous system plays a critical role in painful diabetic neuropathy and sympathetic blocks can be an effective management modality of painful diabetic neuropathy. We concluded that the sympathetic nervous system is a valuable therapeutic target of pharmacological and interventional modalities of treatments in painful diabetic neuropathy patients.
Dental follicle stem cells are a group of cells possessing osteogenic, adipogenetic and neurogenic differentiations, but the specific mechanism underlying the multilineage differentiation remains still unclear. Great attention has been paid to bone morphogenetic protein-9 (BMP-9) due to its potent osteogenic activity. In the present study, rat dental follicle stem cells were isolated and purified, and cells of passage 3 underwent adenovirus mediated BMP-9 gene transfection to prepare dental follicle stem cells with stable BMP-9 expression. Detection of alkaline phosphatase (ALP) and calcium deposition showed dental follicle stem cells transfected with BMP-9 gene could significantly promote the osteogenesis. In addition, SB203580 and PD98059 were employed to block the p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK1/2), respectively. Detection of ALP and calcium deposition revealed the BMP-9 induced osteogenic differentiation of dental follicle stem cells depended on MAPK signaling pathway.
Dental follicle stem cells; rat recombinant adenovirus transfection; bone morphogenetic protein-9; osteogenic differentiation; signaling pathway.
Modification of Notch receptors by O-linked fucose and its further elongation by the Fringe family of glycosyltransferase has been shown to be important for Notch signaling activation. Our recent studies disclose a myeloproliferative phenotype, hematopoietic stem cell (HSC) dysfunction, and abnormal Notch signaling in mice deficient in FX, which is required for fucosylation of a number of proteins including Notch. The purpose of this study is to assess the self-renewal and stem cell niche features of fucose-deficient HSCs.
STUDY DESIGN AND METHODS
Homeostasis and maintenance of HSCs derived from FX-/- mice were studied by serial bone marrow transplantation, homing assay, and cell cycle analysis. Two-photon intravital microscopy was performed to visualize and compare the in vivo marrow niche occupancy by fucose-deficient and wild type (WT) HSCs.
Marrow progenitors from FX-/- mice had mild homing defects that could be partially prevented by exogenous fucose supplementation. Fucose-deficient HSCs from FX-/- mice displayed decreased self-renewal capability compared with the WT controls. This is accompanied with their increased cell cycling activity and suppressed Notch ligand binding. When tracked in vivo by 2-photon intravital imaging, the fucose-deficient HSCs were found localized further from the endosteum of the calvarium marrow than the WT HSCs.
The current reported aberrant niche occupancy by HSCs from FX-/- mice, in the context of a faulty blood lineage homeostasis and HSC dysfunction in mice expressing Notch receptors deficient in O-fucosylation, suggests that fucosylation modified Notch receptor may represent a novel extrinsic regulator for HSC engraftment and HSC niche maintenance.
Autoinflammatory diseases are characterized by seemingly unprovoked episodes of inflammation, without high titers of autoantibodies or antigen-specific T cells, and derive from genetic variants of the innate immune system. This study characterized a cohort of patients with similar phenotypes and nucleotide oligomerization domain 2 (NOD2) gene mutations.
Diagnostically challenging patients with the following clinical and genetic characteristics were prospectively studied between January 2009 and April 2011: periodic fever, dermatitis, polyarthritis, serositis, negative serum autoantibodies and additional positive NOD2 IVS8+158 gene mutation. Genetic testing for gene mutations of NOD2, tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS) and familial Mediterranean fever (FMF) was performed.
All seven patients with the disease were Caucasians, with four being male. The mean age at disease onset was 40.7 years and disease duration was 3.2 years. These patients characteristically presented with periodic fever, dermatitis and inflammatory polyarthritis. There were gastrointestinal symptoms in three patients, granulomas of the skin and gut in two, and recurrent chest pain in two, with one having pleuritis and pericarditis. Three patients had sicca-like symptoms. Five patients had increased acute phase reactants. All seven patients had negative tests for autoantibodies but carried the NOD2 gene mutation IVS8+158 with four having concurrent R702W mutation.
Our cohort may represent a new disease category of autoinflammatory disease with characteristic clinical phenotypes and genotypes. It may somewhat resemble pediatric Blau's syndrome.
Duchenne muscular dystrophy (DMD) is the most common genetic muscle disease affecting 1 in 3,500 live male births. It is an X-linked recessive disease caused by a defective dystrophin gene. The disease is characterized by progressive limb weakness, respiratory and cardiac failure and premature death. Fibrosis is a prominent pathological feature of muscle biopsies from patients with DMD. It directly causes muscle dysfunction and contributes to the lethal DMD phenotype. Although gene therapy and cell therapy may ultimately provide a cure for DMD, currently the disease is devastating, with no effective therapies. Recent studies have demonstrated that ameliorating muscle fibrosis may represent a viable therapeutic approach for DMD. By reducing scar formation, antifibrotic therapies may not only improve muscle function but also enhance muscle regeneration and promote gene and stem cell engraftment. Antifibrotic therapy may serve as a necessary addition to gene and cell therapies to treat DMD in the future. Therefore, understanding cellular and molecular mechanisms underlying muscle fibrogenesis associated with dystrophin deficiency is key to the development of effective antifibrotic therapies for DMD.
Antifibrotic therapy; Duchenne muscular dystrophy; Muscle fibrosis
Muscle fibrosis is a prominent pathological feature that directly causes muscle dysfunction in Duchenne muscular dystrophy (DMD). The DMD mouse models, mdx mice and mdx mice with haploinsufficiency of the utrophin gene (mdx/utrn+/−), display progressive diaphragm fibrosis.
Materials and Methods
We performed unrestrained whole-body plethysmography (WBP) in mdx and mdx/utrn+/− mice, and compared them with wild-type controls.
Respiratory function gauged by respiratory frequency, tidal volume, minute volume, peak inspiratory flow, and peak expiratory flow was significantly impaired in mdx mice. Consistent with more severe diaphragm fibrosis in mdx/utrn+/− mice, respiratory impairment was worse than in mdx mice at 6 months.
WBP is useful in monitoring in vivo respiratory function of mdx and mdx/utrn+/− mice, and it may serve as an outcome measurement of therapies that target diaphragm fibrosis. Mdx/utrn+/− mice may be a better model than mdx mice for testing antifibrotic therapies, especially at a severe stage.
Duchenne muscular dystrophy; mdx mice; diaphragm fibrosis; respiratory function; whole body non-invasive plethysmography
The authors consider the analysis of hierarchical longitudinal functional data based upon a functional principal components approach. In contrast to standard frequentist approaches to selecting the number of principal components, the authors do model averaging using a Bayesian formulation. A relatively straightforward reversible jump Markov Chain Monte Carlo formulation has poor mixing properties and in simulated data often becomes trapped at the wrong number of principal components. In order to overcome this, the authors show how to apply Stochastic Approximation Monte Carlo (SAMC) to this problem, a method that has the potential to explore the entire space and does not become trapped in local extrema. The combination of reversible jump methods and SAMC in hierarchical longitudinal functional data is simplified by a polar coordinate representation of the principal components. The approach is easy to implement and does well in simulated data in determining the distribution of the number of principal components, and in terms of its frequentist estimation properties. Empirical applications are also presented.
Functional data analysis; Hierarchical models; Longitudinal data; Markov chain monte carlo; Principal Components; Stochastic approximation
The connectional and physiological characteristics of the central mesencephalic reticular formation (cMRF) indicate that it participates in gaze control. The cMRF receives projections from the ipsilateral superior colliculus (SC) via collaterals of predorsal bundle axons. These collaterals target cMRF neurons, which in turn project back upon the SC. In the present study, we examined the pattern of connections made by the cMRF reticulotectal projection by injecting the bidirectional neuroanatomical tracer, biotinylated dextran amine (BDA), into the cMRF of macaque monkeys. Anterogradely labeled reticulotectal terminals were found bilaterally in the SC, with an ipsilateral predominance, and were concentrated in the intermediate gray layer (SGI). BDA also retrogradely labeled SC neurons projecting to the cMRF. These labeled tectoreticular cells were located mainly in SGI. Injection site specific differences in the SC labeling pattern were evident, suggesting the lateral cMRF is more heavily connected to the upper sublamina of SGI, whereas the medial cMRF is more heavily connected with the lower sublamina. In view of the known downstream connections of the cMRF and these SC sublaminae, this organization intimates that the cMRF may contain subdivisions specialized to modulate the eye and the head components of gaze changes. In addition, reticulotectal terminals were observed to have close associations with retrogradely labeled tectoreticular cells in the ipsilateral SC, indicating possible synaptic contacts. Thus, the cMRF’s reciprocal connections with the SC suggest this structure plays a role in defining the gaze-related bursting behavior of collicular output neurons.
oculomotor; gaze; saccade; head movement
Hierarchical functional data are widely seen in complex studies where sub-units are nested within units, which in turn are nested within treatment groups. We propose a general framework of functional mixed effects model for such data: within unit and within sub-unit variations are modeled through two separate sets of principal components; the sub-unit level functions are allowed to be correlated. Penalized splines are used to model both the mean functions and the principal components functions, where roughness penalties are used to regularize the spline fit. An EM algorithm is developed to fit the model, while the specific covariance structure of the model is utilized for computational efficiency to avoid storage and inversion of large matrices. Our dimension reduction with principal components provides an effective solution to the difficult tasks of modeling the covariance kernel of a random function and modeling the correlation between functions. The proposed methodology is illustrated using simulations and an empirical data set from a colon carcinogenesis study. Supplemental materials are available online.
Correlated functions; Functional data; Longitudinal data; Mixed effects models; Penalized splines; Principal components; Reduced rank models
In order to evaluate the effects of fatty acids on immune cell membrane structure and function, it is often necessary to maintain cells in culture. However, cell culture conditions typically reverse alterations in polyunsaturated fatty acid (PUFA) composition achieved by dietary lipid manipulation. Therefore, we hypothesized that T-cells from transgenic mice expressing the Caenorhabditis elegans n-3 desaturase (fat-1) gene would be resistant to the culture-induced loss of n-3 PUFA and, therefore, obviate the need to incorporate fatty acids or homologous serum into the medium. CD4+ T-cells were isolated from (i) control wild type (WT) mice fed a safflower oil-n-6 PUFA enriched diet (SAF) devoid of n-3 PUFA, (ii) fat-1 transgenic mice (enriched with endogenous n-3 PUFA) fed a SAF diet, or (iii) WT mice fed a fish oil (FO) based diet enriched in n-3 PUFA. T-cell phospholipids isolated from WT mice fed FO diet (enriched in n-3 PUFA) and fat-1 transgenic mice fed a SAF diet (enriched in n-6 PUFA) were both enriched in n-3 PUFA. As expected, the mol% levels of both n-3 and n-6 PUFA were decreased in cultures of CD4+ T-cells from FO-fed WT mice after 3 d in culture. In contrast, the expression of n-3 desaturase prevented the culture-induced decrease of n-3 PUFA in CD4+ T-cells from the transgenic mice. Carboxyfluorescein succinidyl ester (CFSE) -labeled CD4+ T-cells from fat-1/SAF vs. WT/SAF mice stimulated with anti-CD3 and anti-CD28 for 3 d, exhibited a reduced (P<0.05) number of cell divisions. We conclude that fat-1-containing CD4+ T-cells express a physiologically relevant, n-3 PUFA enriched, membrane fatty acid composition which is resistant to conventional cell culture-induced depletion.
n-3 fatty acid desaturase; Fish oil; Phospholipids; Lymphocyte
To address whether mdx mice with haploinsufficiency of utrophin (mdx/utrn+/-) develop more severe skeletal muscle inflammation and fibrosis than mdx mice, to represent a better model for Duchenne muscular dystrophy (DMD), we performed qualitative and quantitative analysis of skeletal muscle inflammation and fibrosis in mdx and mdx/utrn+/- littermates. Inflammation was significantly worse in mdx/utrn+/- quadriceps at age 3 and 6 months and in mdx/utrn+/- diaphragm at age 3 but not 6 months. Fibrosis was more severe in mdx/utrn+/- diaphragm at 6 months, and at this age, mild fibrosis was noted in quadriceps of mdx/utrn+/- but not mdx mice. The findings indicate that utrophin compensates, although insufficiently, for the effects of dystrophin loss with regard to inflammation and fibrosis of both quadriceps and diaphragm muscles in mdx mice. With more severe muscle dystrophy than mdx mice and a longer life span than utrophin-dystrophin-deficient (dko) mice, mdx/utrn+/- mice provide a better mouse model for testing potential therapies for muscle inflammation and fibrosis associated with DMD.
Duchenne muscular dystrophy; mouse model; mdx; mdx/utrn+/-; inflammation; fibrosis
We propose a modelling framework to study the relationship between two paired longitudinally observed variables. The data for each variable are viewed as smooth curves measured at discrete time-points plus random errors. While the curves for each variable are summarized using a few important principal components, the association of the two longitudinal variables is modelled through the association of the principal component scores. We use penalized splines to model the mean curves and the principal component curves, and cast the proposed model into a mixed-effects model framework for model fitting, prediction and inference. The proposed method can be applied in the difficult case in which the measurement times are irregular and sparse and may differ widely across individuals. Use of functional principal components enhances model interpretation and improves statistical and numerical stability of the parameter estimates.
Some key words: Functional data; Longitudinal data; Mixed-effects model; Penalized spline; Principal component; Reduced-rank model
Amblyopia is the most common cause of visual impairment in children. Early detection of amblyopia and subsequent intervention are vital in preventing visual loss. Understanding the molecular pathogenesis of amblyopia would greatly facilitate development of therapeutic interventions. An animal model of amblyopia induced by monocular vision deprivation has been extensively studied in terms of anatomic and physiologic alterations that affect visual pathways. However, the molecular events underlying these changes are poorly understood. This study aimed to characterize changes of gene expression profiles in the lateral geniculate nucleus (LGN) associated with amblyopia induced by monocular visual deprivation.
Monocular vision deprivation was generated by either opaque dark contact lens or tarsorrhaphy of newborn rhesus monkeys. LGN was harvested at two or four months following induction of vision deprivation. Laser capture microdissection was used to obtain individual LGN layers for total RNA isolation. Linear T7-based in vitro RNA amplification was used to obtain sufficient RNA to conduct DNA microarray studies. The resulting Affymetrix GeneChip Expression data were analyzed using Affymetrix GeneChip Operating Software. Real-time quantitative polymerase chain reaction and in situ hybridization were used to further analyze expression of selected genes.
Using 52,699 microarray probe sets from a Rhesus array, we identified 116 transcripts differentially expressed between deprived and nondeprived parvocellular layers: 45 genes were downregulated and 71 genes were upregulated in deprived parvocellular layers. We also observed substantial changes in deprived magnocellular laminae: 74 transcripts exhibited altered expression, 42 genes were downregulated, and 32 genes were upregulated. The genes identified in this study are involved in many diverse processes, including binding (calcium ion binding, nucleic acid binding, and nucleotide binding), catalytic activity, and signal transducer activity.
There were significant differences in gene expression profiles between deprived and nondeprived parvocellular layers and magnocellular laminae of LGN. These alterations in gene expression may play a critical role in the molecular pathogenesis of amblyopia. The genes identified in this study may provide potential targets for therapeutic intervention of this disease.
Glycoprotein fucosylation enables fringe-dependent modulation of signal transduction by Notch transmembrane receptors, contributes to selectin-dependent leukocyte trafficking, and is faulty in leukocyte adhesion deficiency (LAD) type II, also known as congenital disorder of glycosylation (CDG)-IIc, a rare human disorder characterized by psychomotor defects, developmental abnormalities, and leukocyte adhesion defects. We report here that mice with an induced null mutation in the FX locus, which encodes an enzyme in the de novo pathway for GDP–fucose synthesis, exhibit a virtually complete deficiency of cellular fucosylation, and variable frequency of intrauterine demise determined by parental FX genotype. Live-born FX(−/−) mice exhibit postnatal failure to thrive that is suppressed with a fucose-supplemented diet. FX(−/−) adults suffer from an extreme neutrophilia, myeloproliferation, and absence of leukocyte selectin ligand expression reminiscent of LAD-II/CDG-IIc. Contingent restoration of leukocyte and endothelial selectin ligand expression, general cellular fucosylation, and normal postnatal physiology is achieved by modulating dietary fucose to supply a salvage pathway for GDP–fucose synthesis. Conditional control of fucosylation in FX(−/−) mice identifies cellular fucosylation events as essential concomitants to fertility, early growth and development, and leukocyte adhesion.
selectin; fucosylation; GDP–fucose; LAD-II; CDG-IIc
DNMT2 is a human protein that displays strong sequence similarities
to DNA (cytosine-5)-methyltransferases (m5C MTases) of
both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence
motifs that are conserved among m5C MTases, including
the consensus S-adenosyl-l-methionine-binding
motifs and the active site ProCys dipeptide. DNMT2 has close homologs
in plants, insects and Schizosaccharomyces pombe,
but no related sequence can be found in the genomes of Saccharomyces
cerevisiae or Caenorhabditis elegans. The
crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-l-homocysteine (AdoHcy)
has been determined at 1.8 Å resolution. The structure
of the large domain that contains the sequence motifs involved in
catalysis is remarkably similar to that of M.HhaI,
a confirmed bacterial m5C MTase, and the smaller target
recognition domains of DNMT2 and M.HhaI are also
closely related in overall structure. The small domain of DNMT2
contains three short helices that are not present in M.HhaI.
DNMT2 binds AdoHcy in the same conformation as confirmed m5C
MTases and, while DNMT2 shares all sequence and structural features with
m5C MTases, it has failed to demonstrate detectable transmethylase
activity. We show here that homologs of DNMT2, which are present
in some organisms that are not known to methylate their genomes,
contain a specific target-recognizing sequence motif including an
invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant
complex in vitro. While the biological function of
DNMT2 is not yet known, the strong binding to DNA suggests that
DNMT2 may mark specific sequences in the genome by binding to DNA
through the specific target-recognizing motif.
Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus of the Cardiovirus genus. Certain strains of TMEV may cause a chronic demyelinating disease, which is very similar to multiple sclerosis in humans, associated with a persistent viral infection in the mouse central nervous system (CNS). Other strains of TMEV only cause an acute infection without persistence in the CNS. It has been shown that sialic acid is a receptor moiety only for the persistent TMEV strains and not for the nonpersistent strains. We report the effect of sialylation on cell surface on entry and the complex structure of DA virus, a persistent TMEV, and the receptor moiety mimic, sialyllactose, refined to a resolution of 3.0 Å. The ligand binds to a pocket on the viral surface, composed mainly of the amino acid residues from capsid protein VP2 puff B, in the vicinity of the VP1 loop and VP3 C terminus. The interaction of the receptor moiety with the persistent DA strain provides new understanding for the demyelinating persistent infection in the mouse CNS by TMEV.
Suppression of gap-junctional communication by various protein kinases, growth factors, and oncogenes frequently correlates with enhanced mitogenesis. The oncogene v-src appears to cause acute closure of gap junction channels. Tyr265 in the COOH-terminal tail of connexin 43 (Cx43) has been implicated as a potential target of v-src, although v-src action has also been associated with changes in serine phosphorylation. We have investigated the mechanism of this acute regulation through mutagenesis of Cx43 expressed in Xenopus laevis oocyte pairs. Truncations of the COOH-terminal domain led to an almost complete loss of response of Cx43 to v-src, but this was restored by coexpression of the independent COOH-terminal polypeptide. This suggests a ball and chain gating mechanism, similar to the mechanism proposed for pH gating of Cx43, and K+ channel inactivation. Surprisingly, we found that v-src mediated gating of Cx43 did not require the tyrosine site, but did seem to depend on the presence of two potential SH3 binding domains and the mitogen-activated protein (MAP) kinase phosphorylation sites within them. Further point mutagenesis and pharmacological studies in normal rat kidney (NRK) cells implicated MAP kinase in the gating response to v-src, while the stable binding of v-src to Cx43 (in part mediated by SH3 domains) did not correlate with its ability to mediate channel closure. This suggests a common link between closure of gap junctions by v-src and other mitogens, such as EGF and lysophosphatidic acid (LPA).
intercellular coupling; MAP kinase; phosphorylation; v-src; Xenopus oocytes
Connexins, like true cell adhesion molecules, have extracellular domains that provide strong and specific homophilic, and in some cases, heterophilic interactions between cells. Though the structure of the binding domains of adhesion proteins have been determined, the extracellular domains of connexins, consisting of two loops of ∼34–37 amino acids each, are not easily studied in isolation from the rest of the molecule. As an alternative, we used a novel application of site-directed mutagenesis in which four of the six conserved cysteines in the extracellular loops of connexin 32 were moved individually and in all possible pairwise and some quadruple combinations. This mapping allowed us to deduce that all disulfides form between the two loops of a single connexin, with the first cysteine in one loop connected to the third of the other. Furthermore, the periodicity of movements that produced functional channels indicated that these loops are likely to form antiparallel β sheets. A possible model that could explain how these domains from apposed connexins interact to form a complete channel is discussed.