[Pyroptosis of lens epithelial cells in diabetic cataract].

Objective: To investigate the possible role of pyroptosis of lens epithelial cells (LECs) in the occurrence of diabetic cataract. Methods: Experimental research. A total of 70 cataract lens anterior capsule and aqueous humor samples were obtained from 70 eyes (70 patients) with cataracts in the operation room of Tianjin Medical University Eye Hospital between March 2020 and November 2020. Patients were divided into the non-diabetic and diabetic groups, with 35 patients (35 eyes) in each group. The expressions of Nod like receptor protein 3 (NLRP3), cysteine-aspartic proteases 1 (caspase-1) and Gasdermin D protein (GSDMD) in the lens anterior capsule were detected by Western blotting, real-time quantitative PCR and immunohistochemical staining. Interleukin (IL)-1ß and IL-18 inaqueous humor were detected by enzyme-linked immunosorbent assay (ELISA). The independent sample t-test was used for statistical analysis. Results: The age was (70±9) years in the diabetic group and (71±8) years in the non-diabetic group. There was no significant difference in age and gender distribution between the two groups (both P>0.05). Western blotting analysis showed that the expression levels of NLRP3, caspase-1 and GSDMD protein in the LECs of anterior capsule were 1.11±0.06, 0.95±0.04 and 0.39±0.03 in the diabetic group, significantly higher than those of the non-diabetic group (0.81±0.04, 0.33±0.11 and 0.16±0.04; t=4.38, 5.36, 4.63; all P<0.05). Real-time quantitative PCR showed that the levels of NLRP3, caspase-1 and GSDMD mRNA in the LECs of anterior capsule were 1.98±0.07, 54.36±4.88 and 6.98±1.18 in the diabetic group, significantly higher than those of the non-diabetic group (1.38±0.16, 15.31±1.51 and 2.41±0.95; t=3.49, 7.64, 3.00, all P<0.05). Immunohistochemical staining showed that the average gray values of NLRP3, caspase-1 and GSDMD in the diabetic group were higher than those of the non-diabetic group (all P˂0.01). ELISA showed that the levels of IL-1ß and IL-18 in the aqueous humor were (4.178±0.028) fg/L and (20.983±0.018) fg/L in the diabetic group, significantly higher than those of the non-diabetic group [(4.063±0.017) fg/L and (20.509±0.073) fg/L; t=20.63, 37.21; both P˂0.01]. Conclusion: Caspase-1-mediated pyroptosis of LECs and the release of inflammatory mediators induced by pyroptosis may be involved in the occurrence and development of diabetic cataract.

Patient-centered care factors and access to care: a path analysis using the Andersen behavior model.

OBJECTIVES: Using the Andersen behavioral model, we examined the complex relationships among geographic access to care, financial disadvantage, patient-centered care factors, and access to care outcomes. STUDY DESIGN: This was a retrospective, cross-sectional study of the US civilian non-institutionalized population. METHODS: Our analytic sample included 15,787 US adults aged 18 years or older who had health insurance coverage for a full year in Medical Expenditure Panel Survey 2014-2015. Structural equation modeling was used to determine the associations among usual source of care, travel time to provider, financial disadvantage, patient-centered care factors (perceived interaction with health provider, shared decision-making, and value of health care), and access to care (perceived access to care and unmet need of health services). RESULTS: Our analysis showed that patient-centered care factors were associated with improved perceived access to care (ß = 0.03 to 0.56, P = .002) and reduced unmet needs of health care (ß = -0.03 to -0.17, P = .03 to < .001). Although longer travel time to provider and having financial disadvantage of paying medical bills had negative effects on access to care outcomes, these associations were mediated by patient-centered care quality factors. CONCLUSIONS: Our findings suggest that better patient-centered care factors are associated with enhanced patient access to care. Efforts that focus on improving patient experience could be an effective approach along with coverage expansion to enhance access to quality care.

Activation of BKCa channels via cyclic AMP- and cyclic GMP-dependent protein kinases by eugenosedin-A in rat basilar artery myocytes.

BACKGROUND AND PURPOSE: The study investigated whether eugenosedin-A, a 5-hydroxytryptamine and alpha/beta adrenoceptor antagonist, enhanced delayed-rectifier potassium (K(DR))- or large-conductance Ca(2+)-activated potassium (BK(Ca))-channel activity in basilar artery myocytes through cyclic AMP/GMP-dependent and -independent protein kinases. EXPERIMENTAL APPROACH: Cerebral smooth muscle cells (SMCs) were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K(+)- and Ca(2+)-channel activities. KEY RESULTS: Eugenosedin-A (1 microM) did not affect the K(DR) current but dramatically augmented BK(Ca) channel activity in a concentration-dependent manner. Increased BK(Ca) current was abolished by charybdotoxin (ChTX, 0.1 microM) or iberiotoxin (IbTX, 0.1 microM), but not affected by a small-conductance K(Ca) blocker (apamin, 100 microM). BK(Ca) current activation by eugenosedin-A was significantly inhibited by an adenylate cyclase inhibitor (SQ 22536, 10 microM), a soluble guanylate cyclase inhibitor (ODQ, 10 microM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 microM and Rp-cGMP, 100 microM), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 microM and KT5823, 0.3 microM). Eugenosedin-A reversed the inhibition of BK(Ca) current induced by the protein kinase C activator, phorbol myristyl acetate (PMA, 0.1 microM). Eugenosedin-A also prevented BK(Ca) current inhibition induced by adding PMA, KT5720 and KT5823. Moreover, eugenosedin-A reduced the amplitude of voltage-dependent L-type Ca(2+) current (I(Ca,L)), but without modifying the voltage-dependence of the current. CONCLUSIONS AND IMPLICATIONS: Eugenosedin-A enhanced BK(Ca) currents by stimulating the activity of cyclic nucleotide-dependent protein kinases. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and thereby relax cerebral SMCs.

Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3beta.

Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.

An expression-packaging-processing vector which selects and maintains 7-kb DNA inserts in the blue T4 phage genome.

We have developed an efficient positive-selection vector to insert foreign DNA segments fused to the T4 ipIII gene (encoding internal protein IPIII) into the bacteriophage T4 genome. By using partial deletions of the T4 e gene, which encodes phage lysozyme, lysozyme activity required for plaque formation is used to select plasmid integrants which restore the e gene. In this work, we demonstrate that DNA inserts more than 7.0 kb in length can be incorporated into a T4 genome lacking the alt gene. In addition, the recombinant T4 not only contains a fusion gene driven by the T4 ipIII promoters, but also packages the fusion protein into the T4 capsid due to targeting by the IPIII portion. This expression-packaging-processing system shows that active IPIII::beta Gal fusion reporter protein is produced and packaged during phage infection.

Protection from proteolysis using a T4::T7-RNAP phage expression-packaging-processing system.

DNA coding for bacteriophage T7 RNA polymerase (T7-RNAP) was inserted into a positive selection-vector form of the T4 genome, placing it under the control of bacteriophage T4 ipIII promoters. The recombinant T4::T7-RNAP fusion phage retained infectivity and produced T7-RNAP in infected cells. Fusion genes were constructed by insertion into a plasmid containing an iPIII (encoding internal protein III) target portion and a bacteriophage T7 promoter region. When Escherichia coli cells containing the plasmid were infected with the T4::T7-RNAP re-phage, the bacteria produced fusion protein at high levels. The newly synthesized T4::T7-RNAP re-phage progeny package and process the fusion protein into the phage capsid during head morphogenesis. In this paper, we demonstrate that truncated T4 internal protein IPIII, human IPIII::beta Glo (beta-globin) fusion protein, E. coli IPIII::beta Glo::beta Gal (beta-galactosidase) triple-fusion protein and IPIII::V3 fusion protein (human immunodeficiency virus envelope protein gp120 V3 region) are expressed at high levels by T4::T7-RNAP induction. With IPIII::beta Glo, expression-packaging-processing (EPP) occurs simultaneously with T4::T7-RNAP re-phage infection. We also demonstrate that T4::T7-RNAP re-phage stabilize unstable proteins such as the X90 fragment of beta Gal, thought to be degraded by the lon protease. An unstable 20-kDa fragment of the large subunit of human cytochrome b558, an integral membrane protein in phagocytes, is subject to proteolytic degradation even when produced in the lon-deficient BL21 strain. However, upon induction with T4::T7-RNAP re-phage, the 20-kDa protein is produced intact.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of four human dynamin-like protein variants in brain tumors.

Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have suggested that it is involved in the formation of coated vesicles. In this report, the differential expression of four human dynamin-like protein splice variants (HdynIV-wildtype [WT], -11, -26, and -37) from various brain tumors was identified by reverse transcription/polymerase chain reaction (RT-PCR). One novel variant (HdynIV-11), not described previously, was identified. The four alternatively spliced variants exhibited tissue specificity in normal tissues. The HdynIV-WT was strongly expressed in the brain, whereas HdynIV-37 was expressed in all tissues examined. Moreover, HdynIV-26 was dominant in the liver and apparently overexpressed in all astrocytomas and most meningiomas and adenomas. This report suggests that HdynIV-26 may cause aberrant protein trafficking and alter vesicle formation in brain tumors. Our results also suggest that dynamin-like protein is associated with various brain tumors and, more importantly, that aberrant expression of the HdynIV-26 variant may play a role in brain tumorigenesis.

Rac1 gene mutations in human brain tumours.

AIMS: Rac1 is a member of the Ras superfamily of small GTPase and plays a fundamental role in cytoskeleton reorganization, regulation of gene expression and cell proliferation, and cellular transformation. Though recent studies point to an involvement of rac1 in tumorigenesis, little is known about the alteration of rac1 gene in human brain tumours. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), TA cloning, and DNA sequencing were performed to detect rac1 gene mutations in the surgical specimens of 45 human brain tumours. RESULTS: Twelve of 45 cases had base changes in the rac1 gene. The frequency of rac1 alterations was seven of 18 meningiomas, three of 14 astrocytomas, one of seven pituitary adenomas, and one of four metastatic brain tumours. No mutation was detected in acoustic neurilemomas. The subtypes of seven meningiomas include three meningotheliomatous, two atypical, one transitional and one angioblastic meningioma. Three astrocytomas had rac1 gene mutation, including one grade II, one grade III, and one grade IV astrocytoma. All of single base changes were transitions, five of them being T to C transitions. Sites of rac1 mutation were found in codons 34, 41 (two cases), 42 (two cases), 43, 44, 46 and 58. These mutations are mainly localized in the putative effector-domain of rac1 gene and may enhance the activity of rac1, which increases the survival of brain tumours. CONCLUSION: Our results suggest that rac1 gene may play a role in some brain tumours of divergent histogenesis and that the alterations of rac1 gene may contribute to tumorigenesis and/or metastasis.

Expression and mutation analysis of the p53 gene in astrocytoma.

The role of p53 gene mutations in the formation or progression of human astrocytic tumors is controversial. We studied the distribution pattern of p53 immunoreactivity and analyzed p53 gene mutations to define the significance of p53 gene mutations in astrocytoma tumorigenesis or malignant progression. Twenty-three astrocytic tumors were evaluated with immunohistochemistry, single-strand conformation polymorphism (SSCP) analysis, and sequence analysis. We also searched MEDLINE to collect data on p53 gene mutation frequencies in astrocytic tumors in order to evaluate the association of p53 mutations and tumor grade. Strong immunoreactivity with a diffuse clustering pattern was found in three of five glioblastomas and seven of 12 anaplastic astrocytomas. Three of four low-grade astrocytomas were immunonegative. The p53 immunopositive cells in the only positively staining low-grade astrocytoma in our study appeared sparsely scattered. The results of immunostaining suggested that clonal expansion was associated with astrocytoma progression. Mutations of the p53 gene were detected in four of the 23 astrocytomas (one glioblastoma and three anaplastic astrocytomas). In the genetic data analysis, 76 of 367 astrocytomas had p53 gene mutations. A significantly greater p53 gene mutation frequency was found in anaplastic astrocytomas or glioblastomas than in the low-grade astrocytomas. The results of these immunohistochemical and genetic studies support the view that p53 gene mutation is associated with the malignant progression from low-grade to high-grade astrocytomas rather than with tumor initiation or promotion.

Elimination of false negative results in the two-hybrid system in the phagocyte NADPH oxidase.

The yeast two-hybrid system is finding increased use in the study of interactions between proteins. In this method, two polypeptides are expressed in yeast as fusion proteins to a transcriptional activator DNA-binding domain (bd) and activating domain (ad), respectively. Interaction between the two polypeptides reconstitutes function of a transactivator which controls expression of reporters. The phagocyte NADPH oxidase is a complex of membrane cytochrome b558 (comprised of subunits p22-phox and gp91-phox) and three cytosol proteins (p47-phox, p67-phox, and p21rac) that translocate to membrane and bind to cytochrome b558. This is the first report to demonstrate that two of cytosolic components of cytochrome b558, p47-phox binding to p67-phox each other. We encountered several methodological problems in the two-hybrid system which are the focus of this report.

Bacteriophage T4 expression-packaging-processing vector that encapsidates HIV-type I GP120-V3 fusion protein inside the head.

Bacteriophage T4 as an expression-packaging-processing vector has recently been developed by using the T4 non-essential capsid scaffold protein IPIII(1-3). The IPIII gene was expressed at high level in E. coli from plasmid and was truncated at its C-terminus to permit construction of gene fusion in three different reading frames of EP-16 vector. Regulated higher-expression PPL reading frame vectors were also constructed. Infection of the plasmid-containing bacteria with bacteriophage mutants deleted for the IPIII gene showed that viable phage encapsidated and proteolytically processed the IPIII fusion proteins. An IPIII gene fused to human immunodeficiency virus type I (HIV-1) gp120 loop3 domain (V3) IPIII-V3 fusion gene products has been constructed and was packaged and processed within viable phage particle. The packaged fusion protein Gp120-V3 may be used for production of vaccine in the future.

Prognostic evaluation in supratentorial astrocytic tumors using p53, epidermal growth factor receptor, c-erbB-2 immunostaining.

Molecular pathology may play an important role in predicting the tumor prognosis, particularly p53, epidermal growth factor receptor (EGFR), and c-erbB-2. We investigated six variables (age, sex, histopathological grade, p53, EGFR, and c-erbB-2) to identify the role of such factors in predicting the outcome of patients with supratentorial astrocytic tumors. Thirty-seven tumors were studied including 9 well-differentiated astrocytomas (WHO grade 2), 19 anaplastic astrocytomas (WHO grade 3), and 9 glioblastomas multiforme (WHO grade 4). In univariate analysis, no statistical significance was found for the prognostic value of the sex (p = 0.2188), age (p = 0.5530), p53 immunostain (p = 0.2194), and c-erbB-2 immunostain (p = 0.4203). A significant correlation with the prognosis was found with respect to the histopathological grade (p = 0.0049) and EGFR expression (p = 0.0284). In multivariate analysis, the histopathological grade was shown to be significant independent variable (p = 0.0152). In WHO grade 2 and 3 astrocytomas, expression of p53 or EGFR was associated with poorer patient outcome. In glioblastomas, expression of p53 was also associated with poorer prognosis. Our studies suggested that conventional histological assessment of supratentorial astrocytic tumors remains the best guide to prognosis. Although no statistical significance was found between the immunostains and survival in variant grades of astrocytomas, there was a trend between p53 or EGFR proteins expression and the decrease of survival time.

Clinical significance of distribution patterns of P53 immunoreactivity in astrocytic tumors.

The difference of prognosis in patients with the same WHO grade of astrocytic tumors suggests that such tumors comprise a heterogeneous group in biological behavior. The correlation between p53 immunoreactivity and prognosis has often been reported but remains controversial. From the perspective of clonal expansion of p53 immunopositive cells, the distribution patterns of p53 immunoreactivity can be divided into four types: negative, scattered, focally clustered, and diffusely clustered. The survival rate was evaluated by classifying the p53 immunoreactivity into two groups: the significantly immunopositive patterns (focally and diffusely clustered types) and the significantly immunonegative patterns (negative and scattered types). The survival analysis showed a highly significant difference between these two patterns within the same WHO grade of astrocytic tumors (p = 0.0185). Our studies demonstrate that the distribution patterns of p53 immunoreactivity, which reflect the trends of clonal expansion of p53 immunopositive cells, are significantly valuable in predicting the prognosis of patients with the same WHO grade of astrocytic tumors.

The expression of rac1 pseudogene in human tissues and in human brain tumors.

BACKGROUND: Recent studies have demonstrated that Rac is a regulator of cell morphology and growth. Rac1 gene appears to have involvement in tumorigenesis and metastatic potential. In our previous study of rac1 gene in 45 human brain tumors, rac1 pseudogene was found. The rac1 pseudogene is an intronless pseudogene and has a similarity of 86% with rac1 nucleotide sequence. The rac1 pseudogene contains 579 nucleotides and only 46 amino acids can be translated. Little is known about the expression of rac1 pseudogene in human tissues or tumors. MATERIALS AND METHODS: The expression of rac1 gene and rac1 pseudogene in different human tissues and brain tumors was investigated by the use of reverse transcriptase-polymerase chain reaction and Northern blotting. RESULTS: The rac1 gene is apparently expressed in these 8 human tissues. The rac1 pseudogene is also apparently expressed in human tissues except for brain tissue. The overexpression of rac1 gene in brain tumors was 8% (2/25) and the overexpression of rac1 pseudogene was 76.9% (20/26). Only two astrocytomas had overexpression of rac1 gene, compared with normal brain tissues. The overexpression of rac1 pseudogene was 6 of 9 in meningiomas, 7 of 9 in astrocytomas, and 7 of 8 in pituitary adenomas. CONCLUSIONS: High frequency of overexpression of rac1 pseudogene was detected in the human brain tumors when compared with that expressed in the normal brain tissues. Our study suggested that the rac1 pseudogene may play an important role of the tumorigenesis of brain.

Rac2 expression and mutation in human brain tumors.

BACKGROUND: Rac1 and Rac2 are interchangeable in NADPH oxidase activation. Rac1 plays an important role in regulating nuclear signalling and in the activation of transcriptional factors that regulate gene expression and cell growth. Our previous study observed mutation in effector region of Rac1 gene in brain tumors. Little is known about the expression and mutation of Rac2 in human brain tumors. METHOD: We examined the expression of Rac2 by using reverse transcriptase-polymerase chain reaction (RT-PCR) and northern blot analysis and the mutation of Rac2 gene by using DNA sequence analysis. FINDINGS: The decreased expression of Rac2 was found in 15 cases (57.7%) including 8 of 10 astrocytomas, 2 of 8 meningiomas, and 5 of 8 pituitary adenomas. Two of 13 cases with decreased expression of Rac2 had gene mutation. Only two of 26 cases had Rac2 overexpression in which no Rac2 gene mutation was found. Four of 8 cases with normal Rac2 expression had Rac2 gene mutation. The site of Rac2 gene mutation had no hot spots and was not concentrated in the effector region. CONCLUSIONS: Our results showed a low frequency of mutation and no hot spots of mutation in Rac2 gene in brain tumors, suggesting a decreased possibility of Rac2 in the brain tumorigenesis. The role of high frequency of decreased Rac2 expression in brain tumors, particularly in malignant astrocytomas, needs further investigations to be elucidated.

Protein folding studies in vivo with a bacteriophage T4 expression-packaging-processing vector that delivers encapsidated fusion proteins into bacteria.

A cloned phage T4 gene which expresses the nonessential capsid scaffold protein IPIII was modified to permit construction and packaging of protein fusions within the capsid. IPIII deletion phage packaged IPIII-beta-galactosidase, IPIII-beta-globin, and IPIII-beta-globin-beta-galactosidase fusion proteins; the latter protein fusion was specifically processed by the T4 gene 21 head morphogenetic proteinase in vivo at a consensus leu(ile)-P1-glu* cleavage site to regenerate beta-galactosidase. Phage inject IPIII-beta-galactosidase protein into bacteria, but less activity is recovered in infections of Escherichia coli dnaK or groEL mutants, suggesting that these host molecular chaperones are required for beta-galactosidase intracellular folding. This expression-packaging-processing (EPP) vector directs protein fusions into capsids for easy detection and purification and permits study of protein delivery and folding in bacteria.

Phagocyte NADPH oxidase p67-phox possesses a novel carboxylterminal binding site for the GTPases Rac2 and Cdc42.

Rac GTPases regulate activation of the phagocyte NADPH oxidase, a multi-component enzyme complex that produces superoxide in response to host infection. GTP-bound Rac binds to the cytosol protein p67-phox enabling it to participate in oxidase assembly. Details of this interaction are poorly understood. Previous studies showed that Rac/p67-phox binding is GTP-dependent and that several Rac1 mutants lost the ability to activate the oxidase even though they still bound p67-phox. Using two hybrid and blot overlay binding methods, we identified a novel binding site in the p67-phox C-terminus that binds Rac1, Rac2, and Cdc42, a related GTPase which does not activate the oxidase. Binding was independent of the GDP/GTP state. We also showed that GTP-Cdc42 binds p67-phox N-terminus similar to GTP-Rac. Therefore, Rac binding to p67-phox is not synonymous with NADPH oxidase activation, and Rac probably participates in other steps of oxidase activation in addition to binding p67-phox.

Genomic organization and molecular characterization of the human ninein gene.

The centrosome plays a key role in the formation of the mitotic spindle, cell polarity, and cell locomotion. Previously we identified a novel centrosomal associated protein hNinein using GSK-3beta as a bait in the yeast two-hybrid assay. In this report, the hNinein genome was found to correspond to 29 exons of genomic sequence on human chromosome 14q22. Promoter analysis predicts that hNinein contains a TATA, two CCAAT, and three GC boxes. The promoter exhibits the following potential transcription factor binding sites: Sp1, p300, and AP-1. In addition, an alternatively spliced isoform, encoded a 2041-amino-acid protein of 237,900 Da, which was designated hNinein-Lm (GenBank AF302773). The hNinein-Lm genome was found to correspond to 28 exons (2'-29). Amino acid sequence comparison with hNinein showed that hNinein-Lm exhibited an EF-hand Ca2+ binding domain in the N-terminus which similar to mouse ninein. Northern blot showed that this hNinein-Lm isoform was expressed more than hNinein in tissues examined. Differential RT-PCR combining Southern blotting also showed that hNinein-Lm is much more abundant compared to hNinein. Two forms of ninein may also imply the status of ninein associated with a pair of the centrioles in the centrosome structure. Furthermore, molecular characterization shows that human ninein is oligomerized at the C-terminal end which overlapped with GSK-3beta binding site, suggesting that oligomerization of ninein may be regulated by GSK-3beta phosphorylation.

Boundary sequences of the NADPH oxidase p67(phox) C-terminal SH3 domain play on its specificity.

SH3 domains are found in many signal transduction proteins where they mediate protein-protein binding by recognizing specific peptides rich in proline. Based on the analysis of sequence alignment data, the NADPH oxidase p67(phox) C-terminal SH3 domain possesses a typical compact beta-barrel consisting of five beta-strands arranged in two antiparallel beta-sheets of three and two beta-strands. Multiple amino acid substitutions were made at beta e and its flanking residues to determine the role of the boundary sequences in binding activity and conformational specificity of the domain. Analysis of amino acid P55 indicated that all mutants were completely abolished in their binding activities. The substitution of F58 with Y58 showed no effect of the binding, whereas substitution with stop codon abolished activity. Furthermore, when amino acid V59 was substituted with stop codon, activity was also completely abolished. Substitution of E60 with stop codon showed no effect of binding. Moreover, our data show that V59 particularly could not be replaced by Leu. Taken together, these data suggest that V59 may not only contribute the exact boundary site but also play on the specificity for protein-protein interactions in phagocyte NADPH oxidase.

Three rat brain alternative splicing dynamin-like protein variants: interaction with the glycogen synthase kinase 3beta and action as a substrate.

Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have shown that it may be involved in the formation of coated vesicles. In this report, three different alternatively spliced dynamin-like protein variants (DLP1-WT, -11, and -37) from rat brain were identified by reverse transcription/polymerase chain reaction (RT-PCR). One novel rat alternatively spliced variant (DLP1-37), not described previously, was identified. We examined the interaction of these three rat brain dynamin-like protein variants with glycogen synthase kinase 3beta (Gsk-3beta) using the yeast two-hybrid screening, in vitro binding assay, and immunoprecipitation analysis. It was found that all three examined rat brain dynamin-like protein variants can bind to Gsk-3beta. Moreover, in vitro kinase (phosphorylation) assay showed that mammalian dynamin-like protein acts as a substrate for glycogen synthase kinase 3beta. These data suggest that Gsk-3beta may participate in a functional role in dynamin-like proteins in vesicle trafficking.