PI3K/AKT signaling determines a dynamic switch between distinct KSRP functions favoring skeletal myogenesis.

Skeletal myogenesis is orchestrated by distinct regulatory signaling pathways, including PI3K/AKT, that ultimately control muscle gene expression. Recently discovered myogenic micro-RNAs (miRNAs) are deeply implicated in muscle biology. Processing of miRNAs from their primary transcripts is emerging as a major step in the control of miRNA levels and might be well suited to be regulated by extracellular signals. Here we report that the RNA binding protein KSRP is required for the correct processing of primary myogenic miRNAs upon PI3K/AKT activation in myoblasts C2C12 and in the course of injury-induced muscle regeneration, as revealed by Ksrp knock-out mice analysis. PI3K/AKT activation regulates in opposite ways two distinct KSRP functions inhibiting its ability to promote decay of myogenin mRNA and activating its ability to favor maturation of myogenic miRNAs. This dynamic regulatory switch eventually contributes to the activation of the myogenic program.

Near-field scanner for moving molecules.

We have fabricated using electron beam nanolithography a fixed slit near-field optical scanning device which uses near-field fluorimetry to achieve 200 nm spatial resolution of objects moving over the slits. We explore the basic physics of operating narrow slits in the waveguide cutoff mode and present data from the passage of extended double-stranded DNA molecules passing over the slits as a first example of how this device can be used to do ultrahigh spatial resolution mapping of long polymers.

Separation of 100-kilobase DNA molecules in 10 seconds.

Long double-stranded DNA molecules were separated in microfabricated hexagonal arrays in less than 1 min, several orders of magnitude faster than by using conventional technology. DNA samples were first concentrated at the entrance to the array in a thin band by entropic focusing. They were then separated by pulsed field electrophoresis. T4 (168.9 kbp) and lambda (48.5 kbp) DNAs could be resolved into two clearly separated bands in approximately 10 s in these experiments. This corresponds to a mass resolution of 6% in 11 min in a 1-cm-long array.

A comparison of methods for yeast identification including CHROMagar Candida, Vitek system YBC and a traditional biochemical method.

BACKGROUND: CHROMagar Candida (CAC) is a new chromogenic medium for the presumptive identification of clinically-important yeast isolates. A yeast biochemical card (YBC), a part of the Vitek system is an automatic method for the identification of clinically-important yeast isolates. We conducted a comparison of these two methods with a traditional biochemical method in order to choose a rapid and accurate technique for yeast identification. METHODS: All yeast isolates were inoculated onto Sabourand dextrose agar (SDA) and CAC, and incubated at 30 degrees C for 48 hours. All isolates were simultaneously tested using traditional biochemical methods and the yeast biochemical card from the Vitek system. RESULTS: We evaluated 235 yeast isolates from clinical specimens, including 89 Candida albicans, 47 Candida tropicalis, 43 Candida glabrata, six Trichosporon beigelii, and five Candida krusei in addition to 45 isolates of other yeast species. Isolates were presumptively identified on the basis of colony color and appearance on CAC medium. These observations were compared with a traditional biochemical yeast-identification method and also with YBC from the Vitek system. For five commonly-isolated species (Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei and Trichosporon beigelii), agreement among the CAC medium, YBC method and traditional biochemical method were 98.9% (187/189), 96.3% (182/189), 100% (189/189), respectively. CONCLUSIONS: From our comparison, the CAC medium is a convenient and economic method to identify five commonly-noted yeast species, and the YBC method warrants a greater cost and requires a longer period of time to obtain reliable results.

Hereditary cyclic neutropenia in the male members of a Chinese family with inverted Y chromosome.

We describe four male members of a Chinese family, including the father and three sons, with hereditary cyclic neutropenia. These patients had all developed cyclic neutropenia in childhood with a cycle of around 21 d. Recurrent mucosa and skin infections with fever had occurred frequently, but gradually decreased in severity on reaching adulthood. Monocytosis was found during the neutrophil nadirs in all four patients. Mildly increased serum immunoglobulin (Ig)A and IgG levels, low levels of serum stem cell factor, as well as decreased sperm count and motility were demonstrated in the two elder sons. Chromosomal analysis showed a pericentric inversion of Y chromosome [46,X, inv(Y)(p11.2;q11.23)] in all of the men. These findings may suggest an association between cyclic neutropenia with oligospermia and inv(Y)(p11.2;q11.23) in this particular family.

An Sp1 binding site involves the transcription of the Fas ligand gene induced by PMA and ionomycin in Jurkat cells.

The transcriptional regulation of the Fas ligand (FasL) gene in Jurkat cells was investigated. We demonstrated that an Sp1 binding site, located between -280 and -275 bp relative to the translational start site (+1) of the FasL gene, was important for the transcription of the FasL gene by deletion and mutation analysis in Jurkat cells after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. Nuclear extract of Jurkat cells formed complexes with the oligonucleotides bearing the Sp1 site within -280 to -275 of the FasL promoter. Apart from the constitutive complexes, a new complex was observed after PMA and ionomycin stimulation. Plasmid containing the Sp1 site sequence with site-directed mutation reduced the FasL promoter activity in driving the expression of reporter luciferase gene expression in transfected Jurkat cells after PMA and ionomycin stimulation. The binding of activated Jurkat cell nuclear extract to the mutated Sp1 binding site of the FasL promoter was ablated. In addition, the oligomer containing the Sp1 site of the FasL promoter could compete with oligomer with conserved Sp1 binding sequence in nuclear protein binding of activated Jurkat cells. The data presented in this study suggest that the transactivation of the FasL promoter via the Sp1 binding sequence (-280 to -275) involves the PMA- and ionomycin-induced expression of the FasL gene.

Sorting biomolecules with microdevices.

Micro- and nanofabrication techniques have provided an unprecedented opportunity to create a designed world in which separation and fractionation technologies which normally occur on the macroscopic scale can be optimized by designing structures which utilize the basic physics of the process, or new processes can be realized by building structures which normally do not exist without external design. Since microfabrication is exceedingly sophisticated in its development, it is possible to design and construct highly creative microdevices which allow one to probe specific aspects of biological objects. We give examples of uses of micro- and nanofabrication which, as opposed to simply shrinking the size of the vessels or tubes used in macroscopic lab environments, utilize our understanding of the physics of the process to take advantage of fabrication technologies.

Sorting by diffusion: an asymmetric obstacle course for continuous molecular separation.

A separation technique employing a microfabricated sieve has been demonstrated by observing the motion of DNA molecules of different size. The sieve consists of a two-dimensional lattice of obstacles whose asymmetric disposition rectifies the Brownian motion of molecules driven through the device, causing them to follow paths that depend on their diffusion coefficient. A nominal 6% resolution by length of DNA molecules in the size range 15-30 kbp may be achieved in a 4-inch (10-cm) silicon wafer. The advantage of this method is that samples can be loaded and sorted continuously, in contrast to the batch mode commonly used in gel electrophoresis.

Sequence analysis of the Gluconobacter oxydans RecA protein and construction of a recA-deficient mutant.

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, and Pseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.

The G11 gene located in the major histocompatibility complex encodes a novel nuclear serine/threonine protein kinase.

Protein kinases are involved in signal transduction pathways and play fundamental roles in the regulation of cell functions. Here we report that the gene G11 located in the human major histocompatibility complex encodes a novel Ser/Thr protein kinase. The G11 gene products of 41.5 and 30 kDa were expressed in insect cells using the baculovirus system and transiently in the mammalian cell line COS-7. It was found that after immunoprecipitation of the G11 polypeptides from recombinant baculovirus-infected insect cell lysates or transfected COS-7 cell lysates the immunoprecipitates contained a Mn2+-dependent protein kinase activity that phosphorylated alpha-casein at Ser/Thr residues and histone at Ser residues. Furthermore, mutation of the ATP-binding site by converting the invariant lysine in the catalytic domain (amino acid 317) to a proline resulted in the complete ablation of the enzyme activity. This was consistent with the observation that the G11 polypeptide can be covalently modified by the reactive ATP analogue 5'-p-fluorosulfonylbenzoyladenosine in the absence of ATP, and that this modification is prevented in the presence of 1 mM ATP, indicating that the kinase domain of the G11 polypeptide is capable of binding ATP. Immunofluorescence staining of transfected COS-7 cells transiently expressing G11 revealed that this novel Ser/Thr protein kinase is localized predominantly in the nucleus.

Pyrimidinoceptor-mediated potentiation of inducible nitric-oxide synthase induction in J774 macrophages. Role of intracellular calcium.

We have shown that, in murine J774 macrophages, binding of UTP to pyrimidinoceptors stimulates phosphoinositide (PI) breakdown and an increase in [Ca2+]i. In this study, UTP modulation of the expression of inducible nitric-oxide synthase (iNOS) was investigated. Although UTP alone had no effect, stimulation of J774 cells with a combination of UTP (10-300 microM) and LPS (0.1-3 microgram/ml) resulted in a potentiated increase in nitrite levels. In parallel, the amount of iNOS protein induced by LPS was also potentiated by UTP treatment. The UTP potentiating effect was attenuated by U73122, suggesting involvement of the downstream signaling pathways of phosphatidylinositide turnover. The tyrosine kinase inhibitor genistein inhibited both the LPS-induced nitrite response and the UTP potentiation. Conversely, two protein kinase C inhibitors, Ro 31-8220 and Go 6976, and a phosphatidylcholine-specific phospholipase C inhibitor, D609, inhibited LPS-stimulated nitrite induction, but did not affect the potentiating effect of UTP, which was also unaffected by pretreatment with phorbol 12-myristate 13-acetate for 8 h. Furthermore, the UTP-induced potentiation was abolished by BAPTA/AM or KN-93 (a selective inhibitor of Ca2+/calmodulin-dependent protein kinase (CaMK)). Nitrite potentiation and iNOS induction were prominent when UTP was added simultaneously with LPS, with the potentiating effect being lost when UTP was added 3 h after treatment with LPS. Pyrrolidinedithiocarbamate (3-30 microM), an inhibitor of NF-kappaB, caused a concentration-dependent reduction in the nitrite response to LPS and UTP. In electrophoretic mobility shift assays, LPS produced marked activation of NF-kappaB and AP-1, which was potentiated by UTP. LPS-induced degradation of IkappaB-alpha as well as the phosphorylation of IkappaB-alpha were also increased by UTP. Moreover, the UTP-potentiated activation of NF-kappaB and AP-1 and the degradation and phosphorylation of IkappaB-alpha were inhibited by KN-93. Taken together, these data demonstrate that nucleotides, especially UTP, can potentiate the LPS-induced activation of NF-kappaB and AP-1 and of iNOS induction via a CaMK -dependent pathway and suggest that the UTP-dependent up-regulation of iNOS may constitute a novel element in the inflammatory process.

Stable DNA transfection of the primitive protozoan pathogen Giardia lamblia.

We have developed a stable DNA transfection vector pRANneo for genetic manipulation of the primitive protozoan Giardia lamblia. pRANneo was constructed by replacing the protein coding region of a Giardia ran gene with a bacterial neomycin phosphotransferase gene (neo). This plasmid was electroporated into G. lamblia, and the transfectants were selected by G418. pRANneo replicated episomally to approximately 80 copies per G. lamblia trophozoite as demonstrated by dot hybridizations, Southern hybridizations and transformations of the DpnI-treated plasmids into Escherichia coli. pRANneo/GDHluc was then constructed by incorporation of a luciferase expression system into pRANneo to persistently express firefly luciferase in G. lamblia under G418 selection. The NEO and luciferase proteins were detected in the transfected G. lamblia cells by Western blottings. The level of luciferase activity and the plasmid copy number correlated with the concentration of G418. Removal of G418 from the transfectant culture resulted in gradual loss of the plasmid and luciferase activity. The stable DNA transfection system should provide a valuable tool for genetic studies of G. lamblia.

Urine lipids in patients with a history of filariasis.

The presence of lipids in postprandial urine was assessed in 116 patients with a history of filariasis and 70 normal individuals using a biochemical autoanalyzer. Urinary triglycerides (TGs) ranging from 10 to 1955 mg/dl were detected in 13 individuals with a history of chyluria, including 3 with TG levels ranging from 233 to 1955 mg/dl and cholesterol levels of 6-35 mg/dl. Three patients who had a filarial history but without chyluria were also found to have urinary TGs (13-15 mg/dl) without detectable cholesterol. Neither TGs nor cholesterol were detected in the urine of normal individuals. Fluctuations in postprandial urine lipid contents were measured by time course determinations of urinary TG and cholesterol in 17 patients with filariasis and a history of chyluria, 16 patients with filariasis and hydrocele and 16 normal individuals. The level of urine lipid excretion was found to increase within 1-4 h postprandially, with urinary TG levels ranging between 7.8 and 1284 mg/h in eight patients and urinary cholesterol levels between 1.2 and 138 mg/h in seven patients with a history of chyluria. To evaluate the origin of the urine lipids in hematochyluria, fish oil containing 360 mg eicosapentaenoic acid (EPA) and 240 mg docosahexaenoic acid (DHA) was prescribed to a patient with hematochyluria. The excretion of EPA and DHA in urine was increased postprandially in the patient, similar to the elevation of urinary TG and cholesterol. The profile of fatty acids from urine samples showed it was of dietary origin. Our results suggest that postprandial urine lipids, especially TG, might be used as markers for the clinical evaluation of chyluria.

Implications of intermediate filament protein phosphorylation.

Intermediate filament (IF) proteins, a large family of tissue specific proteins, undergo several posttranslational modifications, with phosphorylation being the most studied modification. IF protein phosphorylation is highly dynamic and involves the head and/or tail domains of these proteins, which are the domains that impart most of the structural heterogeneity and hence presumed tissue specific functions. Although the function of IF proteins remains poorly understood, several regulatory roles for IF protein phosphorylation have been identified or are emerging. Those roles include filament disassembly and reorganization, solubility, localization within specific cellular domains, association with other cytoplasmic or membrane associated proteins, protection against physiologic stress and mediation of tissue-specific functions. Understanding the mechanistic and functional aspects of IF protein phosphorylation is providing insights not only regarding the function of this modification, but also regarding the function of IF proteins.

Separation and characterization of two related giardiaviruses in the parasitic protozoan Giardia lamblia.

Giardiavirus encapsidates a 6.2-kb double-stranded (ds) RNA within a capsid that consists of a major 100-kDa capsid protein (p100) and a minor 190-kDa protein (p190). In this study, two nonhomologous 6.2-kb ds RNAs cohabiting in Giardia lamblia trophozoites were found to be separately encapsidated into two distinct virions, one (designated GLV[p100]) whose capsid consists of p100 and p190, and the other (designated GLV[p95]) whose capsid consists of a 95-kDa protein (p95) and a minor p190-equivalent protein. Both types of virions were enriched in the membranous fraction of a lysate from virus-infected G. lamblia cells. Separation of these virions was achieved by CsCl gradient centrifugation following osmotic rupture of the viral particles. By these treatments, the 6.2-kb ds RNA was removed from GLV[p100] whereas that in GLV[p95] remained unchanged, and the two 6.2-kb ds RNAs that had been purified by this protocol displayed differential hybridization properties to viral cDNA probes. Western blotting and peptide mapping experiments show that p100 and p95 were closely related proteins, but each had distinct amino acid sequences. Virus purification and pulse-chase experiments show that GLV[p100] was selectively secreted into the medium whereas GLV[p95] remained within the trophozoites of G. lamblia toward the late phase of cell growth. Secretion of GLV[p100] was not inhibited by Brefeldin A. These findings demonstrate the cohabitation of multiple Giardiavirus species in G. lamblia.

Mitotic arrest with anti-microtubule agents or okadaic acid is associated with increased glycoprotein terminal GlcNAc's.

The two major intermediate filament glycoproteins in human simple epithelia are keratins 8 and 18 (K8/18). A dramatic increase in terminal N-acetylglucosamine (GlcNAc) residues in K8/18 was previously noted after arresting cells in G2/M using anti-microtubule agents. Here we use in vitro galactosylation to show that increased terminal GlcNAc's is a general phenomenon that occurs in glycoproteins isolated from nuclear and plasma membrane fractions after cells are arrested in mitosis using colcemid, nocodazole, or okadaic acid. All three agents also resulted in a hyperphosphorylated form of K8 as determined by phosphatase treatment and tryptic phosphopeptide mapping. The altered glycosylation was found to be independent of microtubule disassembly, and was not directly related to the G2/M phase of the cell cycle after aphidicolin synchronization. Staurosporine (1 microM) inhibited K8/18 phosphorylation in okadaic acid- or nocodazole-treated cells, and inhibited the increase in K8/18 glycosylation without inhibiting the increase in terminal GlcNAc's of membrane-associated glycoproteins. In contrast, brefeldin A resulted in a dramatic increase in terminal GlcNAc's of membrane-associated but not intermediate filament proteins. Golgi complex-related staining using anti-beta-COP antibody showed significant fragmentation under conditions associated with altered membrane protein glycosylation. Our results suggest that Golgi disruption may be involved in the observed increase in terminal GlcNAc's of membrane but not intermediate filament glycoproteins. The mechanism of increased glycoprotein terminal GlcNAc's in association with mitotic arrest appears to be distinct for intermediate filaments and membrane-associated proteins, and in the case of intermediate filament proteins, phosphorylation may play an important role. Some of the effects of agents that induce mitotic arrest may be mediated by glycosylation changes.

Identification of a keratin-associated protein that localizes to a membrane compartment.

We describe the characterization of an acidic glycoprotein (molecular mass approximately 85 kDa) that associates with keratin intermediate filaments of 'simple'-type epithelia. Using a number of anti-keratin monoclonal antibodies, the 85 kDa glycoprotein was identified by co-immunoprecipitation with keratin polypeptides 8 and 18 (K8/18) from the human colonic epithelial cell line HT29 and several other epithelial cell lines. This Keratin-Associated Protein (termed KAP85) was readily detected after in vitro galactosylation of K8/18 immunoprecipitates obtained from mitosis-arrested cells. Its solubilization and detection were dependent on the detergent used, and it was barely detected after in vitro galactosylation of asynchronously growing G0/G1-phase cells. Its poor in vitro galactosylation in G0/G1-phase cells is likely a reflection of the lack of available terminal N-acetylglucosamine residues, since it can be labelled to a similar extent in G0/G1- and G2/M-phase cells using NaIO4/NaB3H4. Glycosidase digestion showed that KAP85 contains high mannose and complex oligosaccharides. Fractionation of total cellular K8/18 into soluble and cytoskeletal insoluble pools showed that KAP85 associates exclusively with the cytoskeletal K8/18 pool. Subcellular fractionation showed that KAP85 co-localizes with a plasma-membrane-enriched fraction that includes the transferrin receptor and KS-1 antigen. Our results demonstrate in vitro evidence of a membrane-associated glycoprotein (KAP85) which may serve as an attachment site for filamentous K8/18.

Changes of respiratory chain enzyme activities in growing rat muscle mitochondria.

The activities of three mitochondrial respiratory chain enzymes, namely rotenone sensitive NADH-cytochrome c reductase (NCCR), succinate-cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) in the extensor digitorum longus muscle were determined in Wistar rats, twenty each, at 3, 4, 5, 6, 10 and 26 weeks of age. The activity of NCCR was extremely low from birth up to 10 weeks of age. The activity of SCCR was stable at 64% to 72% during the first 6 weeks of life and increased to 78% of the adult level at 10 weeks of age. The CCO activity was only 52% of the adult level at 3 weeks of age, increased to 78% to 86% during the next 3 weeks and reached 92% at 10 weeks of age which was not statistically different from the adult level. We conclude that the activities of these 3 respiratory chain enzymes, in muscle mitochondria in rats, were low during development and reached the adult levels at various ages. Before the normal values of these enzyme activities can be established in human pediatric population, age-matched control should be used as the reference value for evaluation of mitochondrial myopathy.

A significant soluble keratin fraction in 'simple' epithelial cells. Lack of an apparent phosphorylation and glycosylation role in keratin solubility.

We studied the solubility of keratin polypeptides 8 and 18 (K8/18), which are the predominant intermediate filaments in the human colonic epithelial cell line HT29. We find that asynchronously growing cells (G0/G1 stage of the cell cycle) have a substantial pool of soluble keratin that constitutes approx. 5% of total cellular keratin. This soluble keratin pool was observed after immunoprecipitation of K8/18 from the cytosolic fraction of cells disrupted using three detergent-free methods. Several other cell lines showed a similar significant soluble cytosolic K8/18 pool. Arrest of HT29 cells in G2/M stage of the cell cycle was associated with a concurrent increase in keratin solubility. Comparison of K8/18 obtained from the soluble cytosolic fraction and the insoluble high-speed pellet fraction showed similar levels of phosphorylation and glycosylation and similar tryptic radiolabeled phospho- and glycopeptide patterns. Soluble K8/18 can form characteristic 10 nm filaments in vitro as determined by electron microscopy. Cross-linking of soluble K8/18 followed by immunoprecipitation resulted in dimeric and tetrameric forms, based on migration in SDS-polyacrylamide gels. In addition, cross-linked and native soluble K8/18 showed similar migration on nondenaturing gels and similar sedimentation after sucrose density gradient centrifugation. Our results indicate that simple epithelial keratins are appreciably more soluble than previously recognized. The soluble keratin form is assembly competent and appears to be primarily tetrameric. Although K8/18 solubility was found to increase during mitotic arrest, glycosylation and phosphorylation did not play an obvious role in generating the soluble fraction, suggesting an alternate mechanism for keratin solubility.