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PubMed2002

Functional analysis of stem cells in the adult rat testis.

Adult stem cells maintain several self-renewing systems and processes in the body, including the epidermis, hematopoiesis, intestinal epithelium, and spermatogenesis. However, studies on adult stem cells are hampered by their low numbers, lack of information about morphologic or biochemical characteristics, and absence of functional assays, except for hematopoietic and spermatogonial stem cells. We took advantage of the recently developed spermatogonial transplantation technique to analyze germ line stem cells of the rat testis. The results indicate that the stem cell concentration in rat testes is 9.5-fold higher than that in mouse testes, and spermatogenic colonies derived from rat donor testis cells are 2.75 times larger than mouse-derived colonies by 3 mo after transplantation. Therefore, the extent of spermatogenesis from rat stem cells was 26-fold greater than that from mouse stem cells at the time of recipient testis analysis. Attempts to enrich spermatogonial stem cells in rat testis populations using the experimental cryptorchid procedure were not successful, but selection by attachment to laminin-coated plates resulted in 8.5-fold enrichment. Spermatogonial stem cells are unique among adult stem cells because they pass genetic information to the next generation. The high concentration of stem cells in the rat testis and the rapid expansion of spermatogenesis after transplantation will facilitate studies on stem cell biology and the introduction of genetic modifications into the male germ line. The functional differences between spermatogonial stem cells of rat vs. mouse origin after transplantation suggest that the potential of these cells may vary greatly among species.

Animals↗
PubMed1998

Functional analysis of conserved histidines in ADP-glucose pyrophosphorylase from Escherichia coli.

Two absolutely conserved histidines and a third highly conserved histidine are noted in 11 bacterial and plant ADP-glucose pyrophosphorylases. These histidines were individually mutagenized in the E. coli enzyme to glutamine in order to determine their function. Glutamine mutations at residues 143 and 156 produced functional enzymes in cell extracts with slightly lower than wild-type specific catalytic activities and with same heat stability characteristics of the wild-type enzyme. Substitution of residue 83 with glutamine however produced an enzyme having decreased thermal stability. Additional mutageneses at residue 83 with asparagine, arginine, or aspartate gave rise to enzymes having a progressively decreasing trend in thermal stability. These mutants are more susceptible to proteolysis than wild-type enzyme. Kinetic analysis of H83Q and H83N indicates that histidine 83 is not involved in the catalytic mechanism or in substrate binding but possibly in maintenance of the active catalytic structure.

Base Sequence↗
PubMed2002

Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer.

Hereditary nonpolyposis colon cancer (HNPCC) is associated with malfunction of postreplicative mismatch repair (MMR). While a majority of HNPCC-associated mutations in the MMR genes MLH1, MSH2, or MSH6 genes cause truncations-and thus loss of function--of the respective polypeptides, little is currently known about the biochemical defects associated with nontruncating mutations. We studied the interactions of six MLH1 variants, carrying either missense mutations or in-frame deletions, with normal PMS2 and tested the functionality of these heterodimers of MLH1 and PMS2 (MutL(alpha)) in an in vitro MMR assay. Three MLH1 carboxy-terminal mutations, consisting of internal deletions of exon 16 (amino acids 578-632) or exon 17 (amino acids 633-663), or a missense R659P mutation in exon 17, affected the formation of a functional MutL(alpha). Interestingly, mutations C77R and I107R in the amino-terminal part of MLH1 did not affect its heterodimerization with PMS2. The complexes MLH1(C77R)/PMS2 and MLH1(I107R)/PMS2, however, failed to complement a MMR-deficient extract lacking a functional MutL(alpha). As all these five mutations were identified in typical HNPCC families and produce nonfunctional proteins, they can be considered disease-causing. In contrast, the third amino-terminal mutation S93G did not affect the heterodimerization, and the MLH1(S93G)/PMS2 variant was functional in the in vitro MMR assay, given thus the nature of the HNPCC family in question. Although the missense mutation segregates with the disease, the mean age of onset in the family is unusually high (approximately 65 years).

Adaptor Proteins, Signal Transducing↗
PubMed2003

An expression system for the functional analysis of pheromone genes in the tetrapolar basidiomycete Schizophyllum commune.

The investigation of putative pheromone genes of basidiomycetes has been difficult since the small open reading frames are essentially annotated on the basis of a C-terminal farnesylation signal. In order to identify the functional reading frame, expression of small DNA fragments in the fungus is necessary. The expression system developed in the presented paper allows fusion to the promoter of the tef1 gene encoding the constitutively and highly expressed translation elongation factor EF1alpha. This system has been shown to be functional using an easily selectable gene, ura1. The application to identification of functional pheromone genes has been shown with the newly detected bap2(4) gene. The Bap2(4) pheromone is the first Balpha pheromone gene activating only a single receptor specificity.

Amino Acid Sequence↗
PubMed2001

Functional analysis of linker-scan mutants spanning the -376, -308, -244, and -238 polymorphic sites of the TNF-alpha promoter.

Tumor necrosis factor alpha (TNF-alpha) promoter polymorphisms have been linked to a large number of diseases but studies examining the possible direct functional effects of these polymorphisms have been contradictory. Previous studies compared TNF-alpha promoter constructs containing single nucleotide changes. We have now made a series of mutant constructs in which regions of the TNF-alpha promoter containing suspected functional single nucleotide polymorphisms, including -376, -308, -244 and -238, were replaced by a 10 bp linker scan sequence. These constructs were transiently transfected into the T cell line Jurkat, the B cell line Raji, and the monocytic cell line U937, and tested for basal and induced transcriptional activity. Mutant constructs covering both the -308 and -376 polymorphisms showed no significant differences in either basal or induced transcriptional activity. Constructs covering the -244/-238 region showed a small increase in basal activity in the U937 cell line. These results indicate (i) that the -308 and -376 regions are of no functional relevance for TNF-alpha promoter transcription, and (ii) that the -244/-238 region does not influence transcription in some cell lines but may have some role in transcription in others.

B-Lymphocytes↗
PubMed1996

Separation and functional analysis of eukaryotic DNA topoisomerases by chromatography and electrophoresis.

DNA topoisomerases are enzymes that control DNA topology by cleaving and rejoining DNA strands and passing other DNA strands through the transient gaps. Consequently, these enzymes play a crucial role in the regulation of the physiological function of the genome. Beyond their normal functions, topoisomerases are important cellular targets in the treatment of human cancers. In this review we summarize current protocols for extracting and purifying DNA topoisomerases, and for separating subtypes and isoforms of these enzymes. Furthermore, we discuss methods for measuring the catalytic activity of topoisomerases and for monitoring the molecular effects of topoisomerase-directed antitumor drugs in cell-free assays.

Base Sequence↗
PubMed2004

Medaka as a model system for the characterisation of cell cycle regulators: a functional analysis of Ol-Gadd45gamma during early embryogenesis.

Numerous studies, mostly performed on mammalian cell cultures, have implicated the Gadd45 family of small acidic proteins in cell cycle control (arrest and/or engagement in the apoptotic pathway). We report here the cloning, detailled expression pattern and functional characterisation in embryonic development of Ol-Gadd45gamma, the Oryzias latipes ortholog of mammalian Gadd45gamma. Its expression pattern, notably in the developing brain (optic tectum) strongly suggests that it is involved in cell cycle exit. Gain-of-function experiments (through mRNA injection) slowed down early development, and produced embryos clearly reduced in size, while morpholino knockdowns resulted in small embryos over-sensitive to DNA damage (UV irradiation). We further demonstrated that, following Ol-Gadd45gamma overexpression, cells are proliferation-arrested before both G1/S and G2/M cell cycle checkpoints, while in the MO-Ol-Gadd45 loss-of-function experiments cells are engaged in apoptosis rather than prevented from proliferating. These results show that Ol-Gadd45gamma is likely to play an important role in coordinating cell fate decisions during neurogenesis; they also demonstrate that the medakafish is a promising model to analyse in vivo the developmental control of the cell cycle.

Amino Acid Sequence↗
PubMed1987

ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A1.

We have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S.GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S.GTP gamma S), forms over a period of 10-15 min at 37 degrees C. Both guanosine 5'-O-(2-thiodiphosphate) and GTP block this quasi-permanent activation. Some S.GTP gamma S forms in membranes that are exposed to CF alone and then to GTP gamma S, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S.GTP gamma S dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45 degrees C is decreased by GTP gamma S. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. We suggest that active S interacts directly with the enzymic A1 fragment of cholera toxin and not with any toxin substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed2002

A single-chain antibody/epitope system for functional analysis of protein-protein interactions.

Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Functional analysis of the terminase large subunit, G2P, of Bacillus subtilis bacteriophage SPP1.

The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5'-RCGG downward arrowCW-3' sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at both bona fide 5'-CTATTGCGG downward arrowC-3' sequences within pacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P(i). H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1PDeltaN62 (lacking domain I) stimulate 3.5- and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.

Adenosine Triphosphatases↗
PubMed2005

Functional analysis of the domains in Cox11.

Cox11 is an intrinsic mitochondrial membrane protein essential for the assembly of an active cytochrome c oxidase complex. Cox11 is tethered to the mitochondrial inner membrane by a single transmembrane helix. Domain mapping was carried out to determine the functional segments of the Cox11 protein. The C-terminal 189 residue Cu(I)-binding domain is shown to be exposed within the mitochondrial intermembrane space. This orientation was demonstrated by the proteolytic susceptibility of a C-terminal Myc epitope tag in mitoplasts but not intact mitochondria. Fusion of the N terminus of Cox11 to the matrix ribosomal protein Rsm22 results in a functional protein capable of suppressing the respiratory defect of both Deltacox11 cells and Deltarsm22 cells. The functionality of the fusion protein suggests that the Cox11 N terminus projects into the matrix. The fusion of the C-terminal segment of Cox11 to Rsm22 resembles a naturally occurring fusion of Cox11 in Schizosaccharomyces pombe to a sequence homologous to the Saccharomyces cerevisiae Rsm22. Studies on a series of SCO1/COX11 chimeras reveal that the matrix domain of Cox11 lacks a specific function, whereas the Cu(I) binding/donating function requires the yeast Cox11 sequence. The Cu(I)-binding domain from human Cox11 cannot functionally replace the yeast sequence. The copper domain of Cox11 may be an important docking motif for Cox1 or a Cox1-associated protein.

Amino Acid Motifs↗
PubMed2005

Characterization and functional analysis of the MAL and MPH Loci for maltose utilization in some ale and lager yeast strains.

Maltose and maltotriose are the major sugars in brewer's wort. Brewer's yeasts contain multiple genes for maltose transporters. It is not known which of these express functional transporters. We correlated maltose transport kinetics with the genotypes of some ale and lager yeasts. Maltose transport by two ale strains was strongly inhibited by other alpha-glucosides, suggesting the use of broad substrate specificity transporters, such as Agt1p. Maltose transport by three lager strains was weakly inhibited by other alpha-glucosides, suggesting the use of narrow substrate specificity transporters. Hybridization studies showed that all five strains contained complete MAL1, MAL2, MAL3, and MAL4 loci, except for one ale strain, which lacked a MAL2 locus. All five strains also contained both AGT1 (coding a broad specificity alpha-glucoside transporter) and MAL11 alleles. MPH genes (maltose permease homologues) were present in the lager but not in the ale strains. During growth on maltose, the lager strains expressed AGT1 at low levels and MALx1 genes at high levels, whereas the ale strains expressed AGT1 at high levels and MALx1 genes at low levels. MPHx expression was negligible in all strains. The AGT1 sequences from the ale strains encoded full-length (616 amino acid) polypeptides, but those from both sequenced lager strains encoded truncated (394 amino acid) polypeptides that are unlikely to be functional transporters. Thus, despite the apparently similar genotypes of these ale and lager strains revealed by hybridization, maltose is predominantly carried by AGT1-encoded transporters in the ale strains and by MALx1-encoded transporters in the lager strains.

Base Sequence↗
PubMed2001

Differential expression and functional analysis of Pitx2 isoforms in regulation of heart looping in the chick.

Pitx2, a bicoid-related homeobox gene, plays a crucial role in the left-right axis determination and dextral looping of the vertebrate developing heart. We have examined the differential expression and function of two Pitx2 isoforms (Pitx2a and Pitx2c) that differ in the region 5' to the homeodomain, in early chick embryogenesis. Northern blot and RT-PCR analyses indicated the existence of Pitx2a and Pitx2c but not Pitx2b in the developing chick embryos. In situ hybridization demonstrated a restricted expression of Pitx2c in the left lateral plate mesoderm (LPM), left half of heart tube and head mesoderm, but its absence in the extra-embryonic tissues where vasculogenesis occurs. RT-PCR experiments revealed that Pitx2a is absent in the left LPM, but is present in the head and extra-embryonic mesoderm. However, ectopic expression of either Pitx2c or Pitx2a via retroviral infection to the right LMP equally randomized heart looping direction. Mapping of the transcriptional activation function to the C terminus that is identical in both isoforms explained the similar results obtained by the gain-of-function approach. In contrast, elimination of Pitx2c expression from the left LMP by antisense oligonucleotide resulted in a randomization of heart looping, while treatment of embryos with antisense oligonucleotide specific to Pitx2a failed to generate similar effect. We further constructed RCAS retroviral vectors expressing dominant negative Pitx2 isoforms in which the C-terminal transcriptional activation domain was replaced by the repressor domain of the Drosophila Engrailed protein (En(r)). Ectopic expression of Pitx2c-En(r), but not Pitx2a-En(r), to the left LPM randomized the heart looping. The results thus demonstrate that Pitx2c plays a crucial role in the left-right axis determination and rightward heart looping during chick embryogenesis.

Amino Acid Sequence↗
PubMed1977

Background Interference Procedure and discriminant function analysis in predicting clinically determined categories of learning disability.

There is a need to determine the extent to which the "Background Interference Procedure" as an adjunct to the Bender-Gestalt can account for criterion variance beyond that level predicted by an optimal battery. Discriminant functions empirically classified subjects into clinical categories of learning disability. A reduced battery of intellective and visual-motor predictors generated two significant functions, accounting for 91% of the variance. The first dimension reflected over-all intellectual functioning, the second, psychomotor skills. Empirical classification accurately categorized 71% of all subjects across five criterion groups. The functions efficiently separated the criteria, but the six Background Interference Procedure predictor variables did not improve prediction. Implications include using the Background Interference Procedure for early screening of learning disabilities and employing discriminant functions for data reduction and construct validation of teachers' and judges' ratings.

Affective Symptoms↗
PubMed1987

Transfer function analysis of the autonomic response to respiratory activity during random interval breathing.

We report a new method for the noninvasive characterization of the frequency response of the autonomic nervous system (ANS) in mediating fluctuations in heart rate (HR). The approach entails computation of the transfer function magnitude and phase between instantaneous lung volume and HR. Broad band fluctuations in lung volume were initiated when subjects breathed on cue to a sequence of beeps spaced randomly in time. We studied 10 subjects in both supine and standing positions. The transfer function, averaged among all the subjects, showed systematic differences between the two postures, reflecting the differing frequency responses of the sympathetic and parasympathetic divisions of the ANS.

Adult↗
PubMed2004

Functional analysis of microbial community in phenol-degrading aerobic granules cultivated in SBR.

Phenol-degrading aerobic granules were cultivated in a sequencing batch reactor with an influent phenol concentration of 500 mg l(-1). Eight strains were isolated from aerobic granules to characterize the functional redundancy of the microbial community in the granules. The specific oxygen utilization kinetics show the eight strains possessed different phenol-degrading activities, with half-saturation constants (Ks) ranging from 0.4 to 70.5 mg phenol l(-1). Two isolates belonging to dominant populations expressed differing functions. The first strain was linked to the function of phenol degradation as this strain has the highest phenol-degrading ability among all isolates, while the second strain was linked to the maintenance of the granule structure because of its strong self-flocculation activity. This study could be used to exploit the granule-based system for treating high-strength wastewaters.

Aerobiosis↗
PubMed1981

Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets.

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.

Antibodies↗
PubMed2001

Functional analysis in type Ia group B Streptococcus of a cluster of genes involved in extracellular polysaccharide production by diverse species of streptococci.

Several species of streptococci produce extracellular polysaccharides in the form of secreted exopolysaccharides or cell-associated capsules. Although the biological properties and repeating unit structures of these polysaccharides are diverse, sequence analysis of the genes required for their production has revealed a surprising degree of conservation among five genes found in the capsule gene cluster of each of several polysaccharide-producing streptococci. To determine the function of these conserved genes, we characterized a series of isogenic mutants derived from a wild-type strain of type Ia group B Streptococcus by selectively inactivating each gene. Inactivation of cpsIaE resulted in an acapsular phenotype, consistent with previous work that identified the cpsIaE product as the glycosyltransferase that initiates synthesis of the polysaccharide repeating unit. Mutants in cpsIaA, cpsIaB, cpsIaC, or cpsIaD produced type Ia capsular polysaccharide, but in reduced amounts compared with the wild type. Analysis of the mutant polysaccharides and of capsule gene transcription in the mutant strains provided evidence that cpsIaA encodes a transcriptional activator that regulates expression of the capsule gene operon. Mutants in cpsIaC or cpsIaD produced polysaccharide of reduced molecular size but with an identical repeating unit structure as the wild-type strain. We conclude that CpsA to -D are not required for polysaccharide repeating unit biosynthesis but rather that they direct the coordinated polymerization and export of high molecular weight polysaccharide.

Bacterial Capsules↗