[Multi-gene mutations in ultrasound-guided fine-needle aspiration specimens of thyroid micronodules and diagnostic value in thyroid microcarcinomas using next-generation sequencing].

Objective: To explore the detection of BRAF, RAS, TERT promoter, and TP53 gene mutations in solitary thyroid micronodule (TMN) specimens obtained by ultrasound-guided fine-needle aspiration (US-FNA) using next-generation sequencing (NGS) technology and assess its diagnostic value in thyroid microcarcinomas (TMC). Methods: On-site recruitment of 428 patients with single suspicious TMC who underwent thyroid ultrasound examination, US-FNA, and NGS from September 2018 to July 2021 at Beijing Tongren Hospital affiliated to Capital Medical University. A total of 147 patients were finally included. NGS was used to detect mutations in the BRAF, RAS, TERT promoter, and TP53 genes in the US-FNA specimens. Comparisons were made between patients with and without gene mutations in terms of age, gender, and the maximum diameter of nodules. The diagnostic efficiency of BRAF mutation for TMC was calculated using the receiver operating characteristic (ROC) curve, with postoperative pathology as the gold standard. Results: The age [M (Q1, Q3)] of the 147 patients was 43.0 (32.0, 51.0) years, and 37 were male (25.2%). Among the 147 US-FNA specimens, 97 (66.0%) were detected with BRAF gene mutations, all of which were p.V600E point mutation; 6 (4.1%) were detected with RAS gene mutation, and no TERT promoter or TP53 gene mutations were detected. Postoperative pathology confirmed that 136 cases were TMC, all of which were papillary thyroid microcarcinomas (PTMC); 11 cases (7.5%)were benign. Among 136 TMC samples, BRAF gene mutations were detected in 97 cases (71.3%). There were no statistically significant differences in age, gender, and maximum nodule diameter between patients with and without BRAF gene mutations (all P>0.05). The sensitivity and specificity of BRAF gene mutation in diagnosing TMC were 71.3% and 100.0%, respectively, with an area under the ROC curve (AUC) (95%CI) of 0.857 (0.789-0.925). For nodules classified as Bethesda Ⅲ-Ⅴ, the sensitivity and specificity were 63.0% and 100.0%, respectively, with an AUC (95%CI) of 0.815 (0.680-0.950). Conclusions: NGS technology can successfully detect multiple gene mutations in US-FNA specimens from TMN patients, especially BRAF gene mutation, and BRAF gene mutation has certain value in diagnosing TMC.

[The impact of increased cardiac output induced by dobutamine on cerebral blood flow based on MRI 3D-pcASL technology].

Objective: To investigate the effect of increased cardiac output induced by dobutamine on cerebral blood flow (CBF) in healthy volunteers using magnetic resonance 3D-pseudo-continuous arterial spin labeling technology. Methods: A prospective study was conducted on 48 healthy volunteers recruited by handy sampling from June 2021 to January 2022. Physiological parameters before (at rest state) and after (under stress state) dobutamine-induced increase in cardiac output were analyzed. Quantitative CBF maps were generated by using arterial spin labeling difference imaging and proton density weighted reference image processing, and CBF changes under the rest and stress states were compared. Multivariable logistic regression model was used to analyze factors associated with reduced CBF. Results: A total of 48 subjects were included, with an age [M (Q1, Q3)] of 25.0 (24.0, 28.0) years, including 43 men and 5 women. Compared with the rest state, the CBF in the anterior cerebral artery [(36.2±6.9) vs (34.5±6.5) ml·(100 g)-1·min-1, P=0.006] and the middle cerebral artery perfusion area [(35.8±6.5) vs (34.1±6.4) ml·(100 g)-1·min-1, P=0.006] decreased under the stress state, however there was no statistically significant change in CBF in the posterior cerebral artery and the vertebral-basilar artery perfusion area (all P>0.05). Logistic regression analysis showed that the decrease in CBF in the anterior cerebral artery and middle cerebral artery supply regions during the stress state were correlated with an increase in diastolic blood pressure [OR (95%CI): 0.887 (0.796-0.989) and 0.895 (0.805-0.994), both P<0.05]. Conclusions: Dobutamine-induced increase in cardiac output leads to a decrease in CBF in anterior cerebral circulation but has no effect on posterior circulation. The increase in diastolic blood pressure is associated with decreased CBF under the stress state. Changes in CBF should be considered in the context of increased cardiac output.

Expressed sequence tag analysis of functional genes associated with adventitious rooting in Liriodendron hybrids.

Liriodendron hybrids (Liriodendron chinense x L. tulipifera) are important landscaping and afforestation hardwood trees. To date, little genomic research on adventitious rooting has been reported in these hybrids, as well as in the genus Liriodendron. In the present study, we used adventitious roots to construct the first cDNA library for Liriodendron hybrids. A total of 5176 expressed sequence tags (ESTs) were generated and clustered into 2921 unigenes. Among these unigenes, 2547 had significant homology to the non-redundant protein database representing a wide variety of putative functions. Homologs of these genes regulated many aspects of adventitious rooting, including those for auxin signal transduction and root hair development. Results of quantitative real-time polymerase chain reaction showed that AUX1, IRE, and FB1 were highly expressed in adventitious roots and the expression of AUX1, ARF1, NAC1, RHD1, and IRE increased during the development of adventitious roots. Additionally, 181 simple sequence repeats were identified from 166 ESTs and more than 91.16% of these were dinucleotide and trinucleotide repeats. To the best of our knowledge, the present study reports the identification of the genes associated with adventitious rooting in the genus Liriodendron for the first time and provides a valuable resource for future genomic studies. Expression analysis of selected genes could allow us to identify regulatory genes that may be essential for adventitious rooting.

Development and characterization of novel microsatellite markers for Ginkgo biloba using 454 pyrosequencing.

As a "living fossil" that is used to understand the evolutionary history of seed plants, Ginkgo biloba is a well-known multipurpose tree with edible seeds, medicinal properties, and ornamental value, but little is known about its genetic diversity. Microsatellite, or simple sequence repeat (SSR), markers have proven to be powerful tools for genetic studies of plants. In this study, we isolated 30 novel polymorphic microsatellite loci in G. biloba using 454 pyrosequencing. The characteristics of these loci were tested with 48 cultivars. The number of alleles (NA) per locus ranged from two to seven. The observed (HO) and expected (HE) heterozygosities ranged from 0.000 to 0.750 and from 0.021 to 0.792, with an average of 0.326 and 0.443, respectively. In terms of genetic diversity in the Ginkgo population, NA was 3.300, NE was 2.090, I was 0.782, HO was 0.326, and HE was 0.443. These polymorphic SSRs will be useful for the assessment of population genetic diversity and resource conservation of G. biloba.

[The multicenter study on the registration and follow-up of low anticoagulation therapy for the heart valve operation in China].

OBJECTIVE: To investigate the optimal anticoagulation methods and monitoring strategy for Chinese patients undergoing heart valve replacement, which is potentially quite different from western populations. METHODS: In this multicenter prospective cohort study, the anticoagulation and monitoring strategy data was acquired from 25 773 in-hospital patients in 35 medical centers and 20 519 patients in outpatient clinic in 11 medical centers from January 1st, 2011 to December 31th, 2015. RESULTS: As for in-hospital patients, mean age of study population was (48.6±11.2) years old; main etiology of valve pathology was rheumatic (87.5%) origin among study cohort; 94.8% of study population received mechanical valve implantation; international normalized ratio (INR) monitoring (in all the study centers) and low-intensity anticoagulation strategy (31 hospitals chose target INR range of 1.5-2.5, and actual values of INR among 89.2% of 100 069 in-hospital monitoring samples were 1.5-2.5), with mean actual INR values of 1.84±0.53, and warfarin dosage of (2.82±0.93) mg/d were widely adopted among the study centers; strategies of in-hospital warfarin administration were similar in all the study centers; complication rates of low-intensity anticoagulation strategy were low in severe hemorrhage (0.02%), thrombosis (0.05%), and thromboembolism (0.05%) events, without anticoagulation-related death.As for 18 974 outpatient clinic patients, the follow-up rate was 92.47%, with a total of 30 012 patient-years (Pty). Anticoagulation-related morbidity and mortality rates were 0.67% and 0.15% Pty; major hemorrhage morbidity and mortality rates were 0.25% and 0.13% Pty; thromboembolism morbidity and mortality rates were 0.45% and 0.03% Pty.The mean dosage of warfarin daily dosage was (2.85±1.23) mg/d and INR value was 1.82±0.57.No significant regional difference in the intensity of anticoagulation therapy was noted during the study. CONCLUSIONS: INR can be used as a normalized indicator for intensity of anticoagulation therapy in China.The optimal anticoagulation intensity with INR range from 1.5 to 2.5 is safe and effective for Chinese patients with heart valve replacement, and there is no significant regional difference in the intensity of anticoagulation therapy.

Development of novel chloroplast microsatellite markers for Ginkgo biloba.

Ginkgo biloba is considered to be a living fossil that can be used to understand the ancient evolutionary history of gymnosperms, but little attention has been given to the study of its population genetics, molecular phylogeography, and genetic resources assessment. Chloroplast simple sequence repeat (cpSSR) markers are powerful tools for genetic studies of plants. In this study, a total of 30 perfect cpSSRs of Ginkgo were identified and characterized, including di-, tri, tetra-, penta-, and hexanucleotide repeats. Fifteen of 21 designed primer pairs were successfully amplified to yield specific polymerase chain reaction products from 16 Ginkgo cultivars. Polymorphic cpSSRs were further applied to determine the genetic variation of 116 individuals in 5 populations of G. biloba. The results showed that 24 and 76% genetic variation existed within and among populations of this species, respectively. These polymorphic and monomorphic cpSSR markers can be used to trace the origin and evolutionary history of Ginkgo.

Protective effect of proanthocyanidin against oxidative ovarian damage induced by 3-nitropropionic acid in mice.

Oxidative stress, which poses a threat to reproductive health, causes many serious female reproductive diseases. In this study, we investigated whether proanthocyanidins (PC) have a protective effect against oxidative stress-induced ovarian damage. Forty female ICR mice were randomized into 4 groups: a control group, a control plus PC group, a 3-nitropropionic acid (3-NPA) group, and a 3-NPA plus PC group. An ovarian oxidative stress model induced by 3-NPA was constructed using female ICR mice. After the animals were sacrificed, their ovaries were collected to measure reactive oxygen species (ROS) levels, the activities of superoxide dismutase (SOD) and catalase (CAT), and the mRNA expression levels of relevant granulosa cell apoptosis genes (Bcl-2, Bax, Bim, FasL, and caspase-3). We also conducted a histological evaluation of granulosa cell apoptosis and follicular atresia. The results showed that compared to the 3-NPA group, ROS levels and activities of T-SOD and CAT in the 3-NPA plus PC group were significantly decreased (P < 0.05), while the ratio of Bcl-2 to Bax in the 3-NPA plus PC group were significantly increased (P < 0.05). mRNA expression levels of Bim, FasL, and caspase-3 in the 3-NPA plus PC group were significantly decreased (P < 0.05), and the percentage of atretic follicles and granulosa cell apoptosis in the 3-NPA plus PC group was significantly decreased (P < 0.05). Collectively, these data indicate that PC has significant protective effects against damage induced by oxidative stress in mouse ovaries. The mechanisms of protection may be related to antioxidation and apoptosis reduction.

gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein.

The polymerization of actin filaments is involved in growth, movement, and cell division. It has been shown that actin polymerization is controlled by gelsolin, whose interactions with actin are activated by calcium ion (Ca2+) and inhibited by membrane polyphosphoinositides (PPI). A smaller Ca2(+)- and PPI-regulated protein, gCap39, which has 49% sequence identity with gelsolin, has been identified by cDNA cloning and protein purification. Like gelsolin, gCap39 binds to the fast-growing (+) end of actin filaments. However, gCap39 does not sever actin filaments and can respond to Ca2+ and PPI transients independently, under conditions in which gelsolin is ineffective. The coexistence of gCap39 with gelsolin should allow precise regulation of actin assembly at the leading edge of the cell.

Kaposi sarcoma-associated herpesvirus promotes tumorigenesis by modulating the Hippo pathway.

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the culprit behind the human disease Kaposi sarcoma (KS), an AIDS-defining malignancy. KSHV encodes a viral G-protein-coupled receptor (vGPCR) critical for the initiation and progression of KS. In this study, we identified that YAP/TAZ, two homologous oncoproteins inhibited by the Hippo tumor suppressor pathway, are activated in KSHV-infected cells in vitro, KS-like mouse tumors and clinical human KS specimens. The KSHV-encoded vGPCR acts through Gq/11 and G12/13 to inhibit the Hippo pathway kinases Lats1/2, promoting the activation of YAP/TAZ. Furthermore, depletion of YAP/TAZ blocks vGPCR-induced cell proliferation and tumorigenesis in a xenograft mouse model. The vGPCR-transformed cells are sensitive to pharmacologic inhibition of YAP. Our study establishes a pivotal role of the Hippo pathway in mediating the oncogenic activity of KSHV and development of KS, and also suggests a potential of using YAP inhibitors for KS intervention.

Expression and distribution of adhesion molecule CD44 in healing corneal epithelia.

PURPOSE: To study isoform expression and cellular distribution of CD44, a cell surface glycoprotein thought to be an adhesion molecule in cell-cell and cell-substratum interactions, during corneal epithelial wound healing. METHODS: Reverse transcription-polymerase chain reaction was performed to determine alternatively spliced rat CD44 isoforms. In situ hybridization was carried out on frozen sections of the rat corneas obtained at different time points after epithelial debridement. 35S-Labeled sense and antisense cRNA that recognizes rat CD44 standard form was used as a probe. Immunofluorescence was used to assess expression and localization of CD44 in the rat corneas during reepithelialization. RESULTS: Corneal epithelia contained several alternatively spliced CD44 variants. Four large CD44 variants with inserts V1 through V10, V2 through V10, V3 through V10, and V4 through V10 were differentially expressed in migratory epithelia. The silver grains, indicating CD44 transcripts, started to increase in the epithelial cells surrounding the wound margin 3 hours after wounding and peaked at 18 hours in the basal epithelial cell layers, at which time the epithelia were actively migrating. As the cells began proliferation after wounding, the density of CD44 mRNA label declined but was still significantly higher than that in control specimens. The label returned to basal level as epithelial cells reverted to their normal phenotype. The location of CD44 on cell surfaces during corneal reepithelialization was consistent with the pattern of mRNA production. In the corneas at 18 hours after wounding, CD44 immunoreactivity was elevated in the entire epithelium, from the leading edge to the limbal-corneal border. As happened for the mRNA, the cell surface CD44 declined as cells differentiated to reestablish the multilayered epithelium. CONCLUSIONS: The expression of CD44 correlates with corneal reepithelialization, suggesting that CD44 may be involved in cell-cell interactions that provide adhesive strength for the much-stressed epithelial sheet and in the cell-substratum interactions that mediate cell migration during reepithelialization.

Differential gene expression in healing rat corneal epithelium.

PURPOSE: The authors used and validated a recently developed method, mRNA differential display, to detect and clone genes that are differentially expressed in healing compared to stationary corneal epithelium. METHODS: RNAs from unwounded and 18-hour postwound corneal epithelia were isolated and subjected to mRNA differential display analysis. The generated cDNAs were used as probes in Northern blot analysis and in situ hybridization to confirm their differential expression and to clone longer or full-length cDNAs from a healing corneal epithelial cDNA library. RESULTS: Changes in the pattern of gene expression in healing epithelium, compared with that in stationary cells, were noted. To date, 15 combinations of 5'- and 3'- primers were used with approximately 1500 mRNA species screened. Differential expression of nine mRNA species were observed. These included four known proteins. They are nonmuscle tropomyosin TM-1, cytokeratin K14, small GTP binding protein rab 11, and amyloid beta-A4 precursor-like protein-2. One is a sequence with homology to type II cytokeratin, and four represent genes with sequences that are unreported. The differential expression of five of these genes was confirmed by Northern blot analysis, in situ hybridization, or both. CONCLUSION: mRNA differential display provides a unique and powerful experimental system to study differential gene expression in wound healing and cell migration. Using this system, differential expression of nine genes was observed. Detection of genes differentially expressed in healing epithelium may prompt studies that will define the specific role of each of the proteins in wound healing.

A role for amyloid precursor-like protein 2 in corneal epithelial wound healing.

PURPOSE: The authors previously showed that the mRNA encoding the amyloid precursor-like protein 2 (APLP2) was one of several genes with upregulated expression in healing corneal epithelium. To elucidate the physiological role of APLP2 in corneal epithelial cells, the expression, distribution, and posttranslational modification of APLP2 were investigated. METHODS: Alternative splicing was assessed by reverse transcription-polymerase chain reaction; translational expression and posttranslational modification were assessed by chondroitinase digestion and Western blotting. The differential distribution of APLP2 in corneal epithelium was examined by in situ hybridization and immunohistochemical analyses. RESULTS: In rat corneal epithelium, the majority of APLP2 mRNA generated from two alternative splicing sites was found to contain the exon encoding a Kunitz protease inhibitor domain in the first site but to lack the exon encoding a 12-amino acid insert in the second site. The absence of the 12-amino acid insert indicated that APLP2 could be modified by the addition of a chondroitin sulfate (CS) glycosaminoglycan chain. The CS proteoglycan nature of APLP2 was verified by chondroitinase digestion. After wounding, APLP2 mRNA and polypeptides were increased markedly in the basal epithelial cells that were actively migrating. Furthermore, APLP2 was observed in the denuded wound bed immediately adjacent to the leading edge of migratory cells and under the epithelial sheet after wound closure. CONCLUSIONS: The wound-induced, basal-cell-specific APLP2 expression correlates with epithelial cell migration. The spatial and temporal expression of Kunitz protease inhibitor-containing, CS-modified APLP2 in healing corneal epithelium is consistent with its hypothesized role(s) in mediating reorganization of the extracellular matrix and dynamic cell-matrix adhesion during reepithelialization.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion.

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3 ± 10.7 and 97.6 ± 7.6 vs 18.3 ± 1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

Cloning and characterization of human homologue of Drosophila retinal degeneration B: a candidate gene for degenerative retinal diseases.

Mutations in the Drosophila retinal degeneration B (D-rdgB) gene cause light-enhanced retinal degeneration. Here, we report the isolation of the cDNA encoding human homologue of the D-rdgB and initial characterization of the gene products. Like D-rdgB, the human rdgB homologue (H-rdgB) is a transmembrane protein with the N-terminus sharing high homology to two closely related cytosolic proteins, phosphatidylinositol transfer protein (PITP) alpha and beta, indicating that rdgB like proteins belong to the family of PITP proteins. Using Northern and Western blotting, we demonstrated that the rdgB homologue is expressed in rat retina, olfactory bulb, and brain, but not in nonneuronal tissues. In the rat retina, immunoreactivity of the rdgB homologue was observed in photoreceptors and throughout the inner nuclear and plexiform layers; the strongest staining was in the inner plexiform layer. In the photoreceptor cells, the rdgB homologue was located primarily in the inner segment where sorting and traffic of membranes required for outer segment assembly take place. These data, together with recent findings showing PITPs as on important component of intracellular membrane traffic apparatus in mammalian cells, suggest that rdgB homologue may play a role in photoreceptor membrane renewal and in neurotransmitter release. Furthermore, using somatic hybrid cell hybridization and fluorescence in situ hybridization H-rdgB gene was mapped to human chromosome 11q13, a region known to contain several retinopathy loci, including Best disease and Bardet-Biedl syndrome I. Therefore, H-rdgB gene is an attractive candidate for several inherited retinal degenerative diseases.

Actin-dependent carotenoid droplet dispersion in permeabilized cultured goldfish xanthophores.

Organelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule-dependent organelle translocations, little is known about actin-dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actin-dependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin-permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti-actin, but not by anti-tubulin or anti-intermediate filament proteins, suggesting a dependence on F-actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.

Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the gelsolin family of proteins.

gCap39 is an actin filament end-capping protein which has a threefold repeated domain structure similar to the N-terminal half of gelsolin. However, unlike gelsolin, gCap39 does not sever actin filaments and dissociates completely from filament ends after calcium removal. We have capitalized on these differences to explore the structural basis for actin filament capping, severing, and their regulation. Using truncated gCap39, generated by limited proteolysis or deletion mutagenesis, we found that actin filament capping requires multiple gCap domains, and almost the entire molecule is necessary for optimal activity. gCap39 domain I, like the equivalent domain in gelsolin, contains an actin monomer binding site. gCap39 domains II-III are, however, different from gelsolin in that they do not bind to the side of actin filaments. Since filament side binding is hypothesized to be the first step in severing, lack of side binding may explain why gCap39 does not sever. This is confirmed directly by swapping gCap39 domains II-III for the side-binding gelsolin domains to generate a chimera which severs actin filaments. The chimera is Ca2+ independent in actin filament severing and capping, although gCap39 domain I itself is regulated by Ca2+.

gCap39 is a nuclear and cytoplasmic protein.

gCap39 is a newly identified member of the Ca(2+)- and polyphosphoinositide-modulated gelsolin family of actin binding proteins which is different from gelsolin in several important respects: it caps filament ends, it does not sever filaments, it binds reversibly to actin, it is phosphorylated in vivo, and it is also present in the nucleus. gCap39 and gelsolin coexist in a variety of cells. To better understand the roles of gCap39 and gelsolin, we have compared their relative amounts and intracellular distributions. We found that gCap39 is very abundant in macrophages (accounting for 0.6% of total macrophage proteins), and is present in 12-fold molar excess to gelsolin. Both proteins are highly induced during differentiation of the promyelocytic leukemia cell line into macrophages. gCap39 is less abundant in fibroblasts (0.04% total proteins) and is present in equal molar ratio to gelsolin. The two proteins are colocalized in the cytoplasm, but gCap39 is also found in the nucleus while gelsolin is not. Nuclear gCap39 redistributes throughout the cytoplasm during mitosis and is excluded from regions containing chromosomes. Our results demonstrate that gCap39 is a nuclear and cytoplasmic protein which has unique as well as common functions compared with gelsolin.

Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin.

Gelsolin is an actin filament-severing and -capping protein that has profound effects on actin filament organization and assembly. It is activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We have previously shown that PPI inhibit actin filament severing by the amino-terminal half of gelsolin and hypothesized that this is mediated through inhibition of actin filament side binding (by domains II-III of gelsolin), a requisite first step in severing. In this paper, we report that the subsequent step in severing, which is mediated by an actin monomer binding site located in domain I of gelsolin, is also regulated by PPI. We used deletional mutagenesis and a synthetic peptide to locate the sequence required for high affinity PPI binding in domain I. Our results show that the PPI-binding sequence has a basic charge distribution that is also present in the PPI-regulated actin filament side binding domain, and the two gelsolin PPI-binding sites have similar PPI-binding affinities. In addition, a similar motif is present in several other PPI-binding proteins, including a highly conserved region in the phospholipase C family. We propose that the sequences identified in gelsolin may represent a consensus for PPI binding in a variety of proteins.

Effects of thymosin beta 4 and thymosin beta 10 on actin structures in living cells.

The beta-thymosins are a family of small proteins originally isolated from the thymus. Recently, two of the major mammalian isoforms, thymosin beta 4 (T beta 4) and thymosin beta 10 (T beta 10), are identified as significant actin monomer sequestering proteins which may be involved in regulating actin filament assembly. To study the cellular function of beta-thymosins, we have used isoform-specific antibodies to determine their concentration and intracellular distribution, and examined the effects of inducing overexpression of T beta 4 and T beta 10 on actin filament structures. Immunofluorescence labeling of peritoneal macrophages showed that both beta-thymosins are uniformly distributed within the cytoplasm. cDNA-mediated overexpression of beta-thymosins in CV1 fibroblasts induced extensive loss of phalloidin-stained actin stress fibers. Stress fibers in the cell center were more susceptible than those at the periphery. There was a decrease in the number of focal adhesions, as evidenced by a decrease in discrete vinculin staining and an increase in diffuse vinculin fluorescence. The majority of the transfected cells had normal shape in spite of extensive loss of actin filaments. Occasionally, cells overexpressing beta-thymosin were observed to divide. In these cells, beta-thymosin was excluded from the midbody which contains an actin filament-rich contractile ring. Our results indicate that T beta 4 and T beta 10 are functionally very similar and both are effective regulators of a large subset of actin filaments in living cells.