scholarly journals Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions

2018 ◽  
Vol 293 (43) ◽  
pp. 16635-16646 ◽  
Author(s):  
Christoph Howe ◽  
Felix Ho ◽  
Anja Nenninger ◽  
Patrícia Raleiras ◽  
Karin Stensjö
Keyword(s):  
Dna Binding ◽  
Iron Oxidation ◽  
Gel Filtration ◽  
Biochemical Property ◽  
Biochemical Properties ◽  
Oxidation Catalysis ◽  
Nostoc Punctiforme ◽  
Experimental Conditions ◽  
Specific Expression

DNA-binding proteins from starved cells (Dps, EC: 1.16.3.1) have a variety of different biochemical activities such as DNA-binding, iron sequestration, and H2O2 detoxification. Most bacteria commonly feature one or two Dps enzymes, whereas the cyanobacterium Nostoc punctiforme displays an unusually high number of five Dps proteins (NpDps1–5). Our previous studies have indicated physiological differences, as well as cell-specific expression, among these five proteins. Three of the five NpDps proteins, NpDps1, -2, and -3, were classified as canonical Dps proteins. To further investigate their properties and possible importance for physiological function, here we characterized and compared them in vitro. Nondenaturing PAGE, gel filtration, and dynamic light-scattering experiments disclosed that the three NpDps proteins exist as multimeric protein species in the bacterial cell. We also demonstrate Dps-mediated iron oxidation catalysis in the presence of H2O2. However, no iron oxidation with O2 as the electron acceptor was detected under our experimental conditions. In modeled structures of NpDps1, -2, and -3, protein channels were identified that could serve as the entrance for ferrous iron into the dodecameric structures. Furthermore, we could demonstrate pH-dependent DNA-binding properties for NpDps2 and -3. This study adds critical insights into the functions and stabilities of the three canonical Dps proteins from N. punctiforme and suggests that each of the Dps proteins within this bacterium has a specific biochemical property and function.

scholarly journals Biochemical alterations in collagen IV induced by in vitro glycation

1996 ◽  
Vol 319 (3) ◽  
pp. 699-704 ◽  
Author(s):  
Hans-M. RAABE ◽  
Hilke MOLSEN ◽  
Snjezana-M. MLINARIC ◽  
Yahya AÇIL ◽  
Gernot H. G. SINNECKER ◽  
...  
Keyword(s):  
Molecular Conformation ◽  
Microvascular Complications ◽  
Biochemical Properties ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Matrix Assembly ◽  
Experimental Conditions ◽  
Biochemical Alterations ◽  
Diabetic Microvascular Complications

Non-enzymic interactions of carbohydrates and proteins are a major feature of cumulative modification in basement membranes in the course of diabetic microvascular complications. To evaluate the significance of both glycation and glycoxidation reactions for subsequent alterations of biochemical properties, we examined the effects of in vitro glycation on distinct collagen IV domains under different experimental conditions. The 7 S domain and the major triple-helical domain from human placental collagen IV were incubated for various time intervals up to 14 days at 37 °C in the presence of different concentrations of either glucose or ribose under oxidative and antioxidative conditions. Carbohydrate-induced non-enzymic modification in two collagen IV domains was revealed by increased cross-linking and fluorescence. In addition, these non-enzymic modifications apparently have a major impact on molecular conformation and thermal stability of collagen IV, which in turn might influence both cell-matrix interactions and matrix assembly.

METABOLIC CHANGES IN TESTICULAR CELLS FROM RATS AFTER LONG-TERM EXPOSURE TO 37 °C IN VIVO OR IN VITRO

1980 ◽  
Vol 85 (3) ◽  
pp. 471-479 ◽  
Author(s):  
F. F. G. ROMMERTS ◽  
F. H. DE JONG ◽  
J. A. GROOTEGOED ◽  
H. J. VAN DER MOLEN
Keyword(s):  
Sertoli Cells ◽  
Leydig Cells ◽  
Biochemical Properties ◽  
Effect Of Temperature ◽  
Seminiferous Tubules ◽  
Experimental Conditions ◽  
Testicular Cells ◽  
Old Rats

Biochemical properties of isolated Leydig cells, Sertoli cells and spermatocytes from rat testes have been investigated after in-vivo or in-vitro exposure of these cells to abdominal temperature (37 °C). The rate of production of testosterone and pregnenolone by isolated Leydig cells from cryptorchid and normal testes from mature rats was not different. Production of pregnenolone by mitochondria prepared from cryptorchid testes was 6·7 times higher than production by mitochondria from normal testes. Sertoli cells prepared from immature rats and incubated in vitro at 32 or 37 °C showed, on day 1 of the culture period, an initial twofold increase in the secretion of androgen-binding protein which was absent after 6 days in culture. In contrast, incorporation of [3H]leucine into secreted proteins was stimulated twofold on day 1 as well as by day 6 of culture. Secretion of oestradiol was increased 30-fold by day 6 when compared with the level found on day 1 when cells had been cultured at 37 °C and the increased secretion of oestradiol was maintained for approximately 2 days when the temperature of incubation was decreased to 32 °C Spermatocytes isolated from seminiferous tubules incubated for 20 h at 37 °C were active in the synthesis of RNA. No degeneration of these cells was observed in testes of 25-day-old rats 5 days after experimental cryptorchidism, whereas under similar conditions massive degeneration of spermatocytes was shown in the testes of mature rats. These results suggest that the effects of temperature on the different testicular cells greatly depend on the experimental conditions used to study the effect of temperature.

Dimerization and DNA binding of the Salmonella enterica PhoP response regulator are phosphorylation independent

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 3979-3987 ◽  
Author(s):  
Philippe Perron-Savard ◽  
Gregory De Crescenzo ◽  
Hervé Le Moual
Keyword(s):  
Dna Binding ◽  
Salmonella Enterica ◽  
Response Regulator ◽  
Gel Filtration ◽  
Regulatory System ◽  
Tryptophan Fluorescence ◽  
Biochemical Properties ◽  
Wild Type ◽  
His Tag ◽  
Target Dna

In Salmonella enterica, PhoP is the response regulator of the PhoP/PhoQ two-component regulatory system that controls the expression of various virulence factors in response to external Mg2+. Previous studies have shown that phosphorylation of a PhoP variant with a C-terminal His tag (PhoPHis) enhances dimerization and binding to target DNA. Here, the effect of phosphorylation on the oligomerization and DNA binding properties of both wild-type PhoP (PhoP) and PhoPHis are compared. Gel filtration chromatography showed that PhoP exists as a mixture of monomer and dimer regardless of its phosphorylation state. In contrast, unphosphorylated PhoPHis was mostly monomeric, whereas PhoPHis∼P existed as a mixture of monomer and dimer. By monitoring the tryptophan fluorescence of the proteins and the fluorescence of the probe 1-anilinonaphthalene-8-sulfonic acid bound to them, it was found that PhoP and PhoPHis exhibited different spectral properties. The interaction between PhoP or PhoPHis and the PhoP box of the mgtA promoter was monitored by surface plasmon resonance. Binding of PhoP to the PhoP box was barely influenced by phosphorylation. In contrast, phosphorylation of PhoPHis clearly increased the interaction of PhoPHis with target DNA. Altogether, these data show that a His tag at the C-terminus of PhoP affects its biochemical properties, most likely by affecting its conformation and/or its oligomerization state. More importantly, these results show that wild-type PhoP dimerization and interaction with target DNA are independent of phosphorylation, which is in contrast to the previously proposed model.

Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product

1992 ◽  
Vol 12 (5) ◽  
pp. 1905-1914
Author(s):  
S M Stirdivant ◽  
H E Huber ◽  
D R Patrick ◽  
D Defeo-Jones ◽  
E M McAvoy ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Cell Growth ◽  
Dna Binding ◽  
Gene Product ◽  
Biochemical Property ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Cellular Proteins ◽  
Molar Ratio ◽  
Dna Binding Activity

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.

scholarly journals An amino-terminal c-myc domain required for neoplastic transformation activates transcription.

1990 ◽  
Vol 10 (11) ◽  
pp. 5914-5920 ◽  
Author(s):  
G J Kato ◽  
J Barrett ◽  
M Villa-Garcia ◽  
C V Dang
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Nuclear Dna ◽  
Neoplastic Transformation ◽  
Biochemical Properties ◽  
Regulatory Function ◽  
Ras Gene ◽  
Amino Terminal

The product of the c-myc proto-oncogene is a nuclear phosphoprotein whose normal cellular function has not yet been defined. c-Myc has a number of biochemical properties, however, that suggest that it may function as a potential regulator of gene transcription. Specifically, it is a nuclear DNA-binding protein with a short half-life, a high proline content, segments that are rich in glutamine and acidic residues, and a carboxyl-terminal oligomerization domain containing the leucine zipper and helix-loop-helix motifs that serve as oligomerization domains in known regulators of transcription, such as C/EBP, Jun, Fos, GCN4, MyoD, E12, and E47. In an effort to establish that c-Myc might regulate transcription in vivo, we sought to determine whether regions of the c-Myc protein could activate transcription in an in vitro system. We report here that fusion proteins in which segments of human c-Myc are linked to the DNA-binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene linked to GAL4-binding sites. Three independent activation regions are located between amino acids 1 and 143, a region that has been shown to be required for neoplastic transformation of primary rat embryo cells in cooperation with a mutated ras gene. These results demonstrate that domains of the c-Myc protein can function to regulate transcription in a model system and suggest that alterations of Myc transcriptional regulatory function may lead to neoplastic transformation.

scholarly journals rnrGene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R

2011 ◽  
Vol 77 (22) ◽  
pp. 7896-7904 ◽  
Author(s):  
Shaheen Sulthana ◽  
Purusharth I. Rajyaguru ◽  
Pragya Mittal ◽  
Malay K. Ray
Keyword(s):  
Pseudomonas Syringae ◽  
Divalent Cations ◽  
Biochemical Property ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Rnase R ◽  
Content Type ◽  
Antarctic Bacterium ◽  
The Antarctic

ABSTRACTRNase R is a highly processive, hydrolytic 3′-5′ exoribonuclease belonging to the RNB/RNR superfamily which plays significant roles in RNA metabolism in bacteria. The enzyme was observed to be essential for growth of the psychrophilic Antarctic bacteriumPseudomonas syringaeLz4W at a low temperature. We present results here pertaining to the biochemical properties of RNase R and the RNase R-encoding gene (rnr) locus from this bacterium. By cloning and expressing a His6-tagged form of theP. syringaeRNase R (RNase RPs), we show that the enzyme is active at 0 to 4°C but exhibits optimum activity at ∼25°C. The enzyme is heat labile in nature, losing activity upon incubation at 37°C and above, a hallmark of many psychrophilic enzymes. The enzyme requires divalent cations (Mg2+and Mn2+) for activity, and the activity is higher in 50 to 150 mM KCl when it largely remains as a monomer. On synthetic substrates, RNase RPsexhibited maximum activity on poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded structuredmalE-malFRNA substrates. Analysis of the cleavage products shows that the enzyme, apart from releasing 5′-nucleotide monophosphates by the processive exoribonuclease activity, produces four-nucleotide end products, as opposed to two-nucleotide products, of RNA chain byEscherichia coliRNase R. Interestingly, three ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNase RPsin vitro. The ability of the nonhydrolyzable ATP-γS to inhibit RNase RPsactivity suggests that nucleotide hydrolysis is not required for inhibition. This is the first report on the biochemical property of a psychrophilic RNase R from any bacterium.

scholarly journals New Amylase Inhibitor Present in Corn Seeds Active In Vitro Against Amylase from Fusarium verticillioides

Plant Disease ◽  
2003 ◽  
Vol 87 (3) ◽  
pp. 233-240 ◽  
Author(s):  
Edson L. Z. Figueira ◽  
Alejandro Blanco-Labra ◽  
Antônio Carlos Gerage ◽  
Elisabete Y. S. Ono ◽  
Elizabeth Mendiola-Olaya ◽  
...  
Keyword(s):  
High Performance ◽  
Fusarium Verticillioides ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Human Saliva ◽  
Exchange Chromatography ◽  
Experimental Conditions ◽  
Amylase Inhibitor ◽  
Fusarium Sp

A screening for specific amylase inhibitor levels against amylase from Fusarium verticillioides (Fusarium moniliforme), the most relevant mycotoxigenic fungus in corn, was conducted on 37 corn hybrids. The amylase inhibitor levels in these hybrids ranged from 5.5 to 16.0 amylase inhibitor units per gram of corn (AIU/g) in the MASTER and AG5011 hybrids, respectively. The hybrid with the maximum content of inhibitor was used as the source of this new protein. The inhibitor was partially purified using fractional precipitation, gel filtration on Sephadex G-75 column, high performance liquid chromatography (HPLC) Superose HR 10/30 column, and HPLC anion exchange chromatography, obtaining a 20.7-fold purification. Electrophoresis after denaturing and heating under reductive conditions showed an apparent 23.8 kDa molecular mass and an acidic isoelectric point of 5.4, which differs from previous molecular masses reported for other inhibitors present in corn seeds (14 and 22 kDa). This inhibitor showed activity against amylases from human saliva and pancreas, from the fungi F. verticillioides and Aspergillus flavus, and from the insects Acanthoscelides obtectus, Zabrotes subfasciatus, Tribolium castaneum, and Sitotroga cerealella. The mycoflora found in the corn grain indicated Fusarium sp. as the most prevalent fungi (81.1% of the samples), with a count ranging from 1.5 × 102 to 2.4 × 106 CFU/g of corn. The presence of fumonisins was detected in 21 out of the 37 hybrids studied, ranging from 0.05 to 2.67 μg of FB per gram of corn. No correlation could be established between this amylase inhibitor level in the corn seeds and the presence of Fusarium sp. or with the fumonisin content under the experimental conditions of the test.

scholarly journals Insect cardioactive peptides. I. Distribution and molecular characteristics of two cardioacceleratory peptides in the tobacco hawkmoth, Manduca sexta

1985 ◽  
Vol 114 (1) ◽  
pp. 365-379 ◽  
Author(s):  
N. J. Tublitz ◽  
J. W. Truman
Keyword(s):  
Manduca Sexta ◽  
Isolated Heart ◽  
Gel Filtration ◽  
Biochemical Properties ◽  
Molecular Characteristics ◽  
Molecular Weights ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Pharate Adult

Using an in vitro heart bioassay, the pharmacological and biochemical properties of two cardioactive peptides derived from neural tissues of the moth, Manduca sexta, were analysed. Gel filtration of ventral nerve cords (VNC) from pharate adults identified two cardioacceleratory peptides (CAP1 and CAP2) with apparent molecular weights of 1000 and 500 Da, respectively. Both CAPs were localized to the abdominal perivisceral organs, the major neurohaemal release sites in the insect VNC. Pulse application of CAP1 or CAP2 on the in vitro Manduca heart produced a dose-dependent increase in rate but had no effect on beat amplitude. The threshold dose for the action of each peptide on the isolated heart bioassay was less than 0.05 abdominal nerve cord equivalents. Both CAPs were present in the pharate adult VNC of several other Lepidopteran species. Neither CAP1 nor CAP2 was detected in the prepupal VNC of Manduca sexta.

scholarly journals Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

1992 ◽  
Vol 12 (5) ◽  
pp. 1905-1914 ◽  
Author(s):  
S M Stirdivant ◽  
H E Huber ◽  
D R Patrick ◽  
D Defeo-Jones ◽  
E M McAvoy ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Cell Growth ◽  
Dna Binding ◽  
Gene Product ◽  
Biochemical Property ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Cellular Proteins ◽  
Molar Ratio ◽  
Dna Binding Activity

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.

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