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In the present paper we investigated the role played by apoptosis during oogenesis in the cartilaginous fish Torpedo marmorata. TEM, TUNEL and immunohistochemical techniques were employed to specifically reveal morphological and biochemical hallmarks of apoptosis in specimens from birth to sexual maturity. Data obtained demonstrate that apoptosis occurs in prefollicular oocyte selection, in maintaining the homeostasis of granulosa in healthy growing oocyte and in resorbing atretic follicles. In this respect, the involvement of apoptosis in Torpedo marmorata oogenesis closely parallels that found in mammals, thus confirming that strategies of germ cell selection among vertebrates have been evolutionarily preserved.
Mol Reprod Dev 2003 Sep
PMID:Role of apoptosis and Fas/FasL system in the oogenesis of the spotted ray Torpedo marmorata. 1287 99

Two allosteric enzymes have been created by the covalent linkage of non-interacting, monomeric proteins with the prerequisite effector-binding and catalytic functionalities, respectively. This was achieved through a combinatorial process called random domain insertion. The fragment of the TEM-1 beta-lactamase gene coding for the mature protein lacking its signal sequence was randomly inserted into the Escherichia coli maltose-binding protein (MBP) gene to create a domain insertion library. This library's diversity derived both from the site of insertion and from a distribution of tandem duplications or deletions of a portion of the MBP gene at the insertion site. From a library of approximately 2 x 10(4) in-frame fusions, approximately 800 library members conferred a phenotype to E.coli cells that was consistent with the presence of bifunctional fusions that could hydrolyze ampicillin and transport maltose in E.coli. Partial screening of this bifunctional sublibrary resulted in the identification of two enzymes in which the presence of maltose modulated the rate of nitrocefin hydrolysis. For one of these enzymes, the presence of maltose increased k(cat) by 70% and k(cat)/K(m) by 80% and resulted in kinetic parameters that were almost identical to TEM-1 beta-lactamase. Such an increase in activity was only observed with maltooligosaccharides whose binding to MBP is known to induce a conformational change. Modulation of the rate of nitrocefin hydrolysis could be detected at maltose concentrations less than 1 microM. Intrinsic protein fluorescence studies were consistent with a conformational change being responsible for the modulation of activity.
J Mol Biol 2004 Feb 06
PMID:Creation of an allosteric enzyme by domain insertion. 1474 Dec 21

The polystyrene-acrylic acid (PS-AA) nanoparticles have been prepared by ultrasonic polymerization, characterized by FT-IR and TEM. It is the first report on the determination of proteins with macromolecules nanoparticles of PS-AA by resonance light-scattering (RLS). At pH 6.9, the RLS of macromolecules nanoparticles of PS-AA can be enhanced by proteins. Based on this, a novel quantitative assay of proteins at the nanogram levels has been proposed. At pH 6.9, the RLS signals of PS-AA were greatly enhanced by proteins in the region of 250-700 nm characterized by the peak at 342 nm. Under optimal conditions, the linear ranges of the calibration curves were 0.02-11.0 microgml-1, 0.04-10.0 microgml-1 and 0.03-10.0 microgml-1 for gamma-globulin (gamma-IgG), bovine serum albumin (BSA) and human serum albumin (HSA), respectively. The detection limits were 16.0 ngml-1, 19.0 ngml-1, and 15.0 ngml-1 for gamma-IgG, BSA and HSA, respectively. The method has been applied to the analysis of total proteins in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital, which indicates that the method presented here is not only sensitive, simple, but also reliable and suitable for practical application.
Spectrochim Acta A Mol Biomol Spectrosc 2004 Mar
PMID:Quantitative determination of proteins at nanogram levels by the resonance light-scattering technique with macromolecules nanoparticles of PS-AA. 1503 83

The chromophoric spin-label substrate 6-N-[3-(2,2,5,5-tetramethyl-1-oxypyrrolin-3-yl)-propen-2-oyl]penicillanic acid (SLPPEN) was synthesized by acylation of 6-aminopenicillanic acid with the acid chloride of 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)-2-propenoic acid and characterized by physical methods. By application of angle-selected electron nuclear double resonance (ENDOR), we have determined the molecular structure of SLPPEN in solution. SLPPEN exhibited UV absorption properties that allowed accurate monitoring of the kinetics of its enzyme-catalyzed hydrolysis. The maximum value of the (substrate-product) difference extinction coefficient was 2824 M(-1) cm(-1) at 275 nm compared to 670 M(-1) cm(-1) at 232 nm for SLPEN [J. Am. Chem. Soc. 117 (1995) 6739]. For SLPPEN, the steady-state kinetic parameters kcat and kcat/KM, determined under initial velocity conditions, were 637 +/- 36 s(-1) and 13.8 +/- 1.4 x 10(6) M(-1) s(-1), respectively, for hydrolysis catalyzed by TEM-1 beta-lactamase of E. coli, and 0.5 +/- 0.04 s(-1) and 3.9 +/- 0.4 x 10(4) M(-1) s(-1) for hydrolysis catalyzed by the beta-lactamase of Enterobacter cloacae P99. We have also observed "burst kinetics" for the hydrolysis of SLPPEN with P99 beta-lactamase, indicative of formation of an acylenzyme reaction intermediate. In DMSO:H2O (30:70, v:v) cryosolvent mixtures buffered to pH* 7.0, the half-life of the acylenzyme intermediate formed with the P99 enzyme at -5 degrees C was > or = 3 min, suitable for optical characterization. The observation of burst kinetics in the hydrolysis of SLPPEN catalyzed by P99 beta-lactamase suggests that this chromophoric spin-labeled substrate is differentially sensitive to active site interactions underlying the cephalosporinase and penicillinase reactivity of this class C enzyme.
Spectrochim Acta A Mol Biomol Spectrosc 2004 May
PMID:Chromophoric spin-labeled beta-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C beta-lactamases. 1513 25

The nanometer-sized ZnO was prepared through the sol-gel method. Its average particle diameter, determined by TEM, was 20-30 nm. The specific surface area was determined to be 22 m2 g(-1) by BET. The photodegradation mechanism of Rhodamine dyes on nanometer-sized ZnO was studied by dynamic molecular spectra, and the results showed that the photodegradation of Rhodamine dyes obeyed the rules of a pseudo first-order kinetic reaction. The rate constant k of the degradation of Rhodamine B (RB) and butyl-Rhodamine (BR) were 0.0128 and 0.0154 min(-1), respectively, and the half period t(1/2) were 60 and 52 min, respectively. The photodegradation reaction conditions were optimized. After intermixing with silver, the photodegradation efficiency was greatly improved. A life-span test showed that nanometer-sized ZnO had a long life-span.
Spectrochim Acta A Mol Biomol Spectrosc 2004 Jun
PMID:Studies on the photodegradation of Rhodamine dyes on nanometer-sized zinc oxide. 1514 6

Tumour endothelial marker-8 (TEM-8) has been found to be selectively upregulated in tumour-associated endothelial cells and is implicated in tumour specific angiogenesis. Specific factors, indigenous to tissues and tumours that regulate the TEM-8 mechanism in angiogenesis are not defined. We report for the first time that interleukin-1beta induces the expression of TEM-8 in endothelial cells. Human vascular endothelial cells (HECV), which strongly express IL-1beta receptor (as revealed by RT-PCR, Western blotting), increased the level of TEM-8 expression following stimulation with IL-1beta (as revealed by conventional and quantitative RT-PCR). Using a newly developed antibody to human TEM-8, we have further demonstrated that IL-1beta significantly raised the level of TEM-8 at the protein level, as revealed by Western blotting. In vitro tubule forming assay, revealed that IL-1beta significantly induced the formation of capillary-like tubules from the HECV cells, accompanied by an increase in TEM-8 expression. It is concluded that IL-1beta is a powerful regulator of the expression of TEM-8 in vascular endothelial cells. Our results suggest an important pathway through which IL-1beta regulates tumour-associated angiogenesis.
Int J Mol Med 2004 Jul
PMID:Upregulation of tumour endothelial marker-8 by interleukin-1beta and its impact in IL-1beta induced angiogenesis. 1520 19

The internally fertilizing primitive frog Ascaphus truei (family Ascaphidae) from the Pacific Northwest is the only frog with an intromittent organ. The more advanced neobatrachian frog Eleutherodactylus coqui (family Leptodactylidae) from Puerto Rico has secondarily acquired internal fertilization but mates by cloacal apposition. Nonetheless, both frogs have introsperm with an elongated head containing highly condensed chromatin. Characterization of sperm nuclear basic proteins (SNBPs) in E. coqui by acid-urea polyacrylamide gel electrophoresis indicates that, as in A. truei, testes from a single animal contain several protamines. Amino acid analysis indicates a composition for the most rapidly moving protamine of each species as follows: in E. coqui, ARG (35.6 mol %) + LYS (3.8 mol %) + HIS (7.6 mol %) = 47 mol % total basic residues and in A. truei, ARG (42.1 mol %) + LYS (11.1 mol %) = 53.2 mol % total basic residues. Transmission electron microscopy shows that E. coqui introsperm, like those in A. truei, are elongate with highly condensed chromatin. However, E. coqui introsperm lacks an axial perforatorium that extends into an endonuclear canal. These morphological features are plesiomorphic (primitive) and shared by A. truei with urodeles and basal amniotes (Jamieson et al. (1993) Herpetologica 49:52-65). In E. coqui introsperm, the nucleoprotein complex has a cross-sectional axis of 420 + 20 angstroms and shows a knobby chromatin structural organization in TEM. The presence of arginine-enriched protamines in both a basal anuran like the ascaphid A. truei and a more advanced neobatrachian like the leptodactylid E. coqui supports the hypothesis that internal fertilization acts as a constraint on the range of SNBP diversity in animals.
Mol Reprod Dev 2005 Mar
PMID:Protamines in the internally fertilizing neobatrachian frog Eleutherodactylus coqui. 1569 90

Characterization of antimicrobial resistance and virulence gene profiles provides important information on the potential pathogenicity of bacteria. This information can be used to facilitate prompt and effective treatment of bacterial infections. We developed and tested a PCR-based microarray platform for detecting virulence and antimicrobial resistance genes in Salmonella serovars and Escherichia coli. Twelve Salmonella and seven E. coli isolates were screened for the presence of 25 virulence and 23 antimicrobial resistance genes. All S. Typhimurium DT104 isolates harbored virulence plasmids. E. coli O157:H7 isolates possessed virulence genes typical of enterohemorrhagic E. coli (EHEC), whereas E. coli O126 isolates contained virulence genes characteristic of enteropathogenic E. coli (EPEC) and E. coli O111, O78 and O147 isolates had virulence genes characteristic of enterotoxigenic E. coli (ETEC). Correlation between antimicrobial resistance phenotype and genotype was observed for each isolate. The aadA, tetA, and sulI genes were most commonly detected in bacteria resistant to streptomycin, tetracycline and sulfonamide, respectively. All isolates exhibiting resistance to third generation cephalosporins harbored the bla(CMY-2) and bla(TEM-1) genes. Microarray analysis is an effective method to rapidly screen Salmonella and E. coli for multiple antimicrobial resistance and virulence genes.
Mol Cell Probes 2005 Jun
PMID:A DNA microarray for identification of virulence and antimicrobial resistance genes in Salmonella serovars and Escherichia coli. 1579 20

Extended spectrum beta-lactamases (ESBLs) confer bacterial resistance to third-generation cephalosporins, such as cefotaxime and ceftazidime, increasing hospital mortality rates. Whereas these antibiotics are almost impervious to classic beta-lactamases, such as TEM-1, ESBLs have one to four orders greater activity against them. The origins of this activity have been widely studied for the TEM and SHV-type ESBLs, but have received less attention for the CTX-M beta-lactamases, an emerging family that is now the dominant ESBL in several regions. To understand how CTX-M beta-lactamases achieve their remarkable activity, biophysical and structural studies were undertaken. Using reversible, two-state thermal denaturation, it was found that as these enzymes evolve a broader substrate range, they sacrifice stability. Thus, the mutant enzyme CTX-M-16 is eightfold more active against ceftazidime than the pseudo-wild-type CTX-M-14 but is 1.9 kcal/mol less stable. This is consistent with a "stability-activity tradeoff," similar to that observed in the evolution of other resistance enzymes. To investigate the structural basis of enzyme activity and stability, the structures of four CTX-M enzymes were determined by X-ray crystallography. The structures of CTX-M-14, CTX-M-27, CTX-M-9 and CTX-M-16 were determined to 1.10 Angstroms, 1.20 Angstroms, 0.98 Angstroms and 1.74 Angstroms resolution, respectively. The enzyme active sites resemble those of the narrow-spectrum TEM-1 and SHV-1, and not the enlarged sites typical of ESBL mutants such as TEM-52 and TEM-64. Instead, point substitutions leading to specific interactions may be responsible for the improved activity against ceftazidime and cefotaxime, consistent with observations first made for the related Toho-1 enzyme. The broadened substrate range of CTX-M-16 may result from coupled defects in the enzyme's B3 strand, which lines the active site. Substitutions Val231-->Ala and Asp240-->Gly, which convert CTX-M-14 into CTX-M-16, occur at either end of this strand. These defects appear to increase the mobility of B3 based on anisotropic B-factor analyses at ultrahigh resolution, consistent with stability loss and activity gain. The unusually high resolution of these structures that makes such analyses possible also makes them good templates for inhibitor discovery.
J Mol Biol 2005 Apr 29
PMID:Atomic resolution structures of CTX-M beta-lactamases: extended spectrum activities from increased mobility and decreased stability. 1581 73

A rapid, high-throughput bacterial mutagenicity test system has been developed (MutaGen test) that detects reversions of inactivating base-pair substitutions and frameshifts in a TEM-1 class A beta-lactamase (ampicillinase) gene. To quickly and sensitively detect mutagens, the system utilises a series of plasmids that contain the mutated ampicillinase gene and the mucAB operon. Inactivating mutations in the ampicillinase gene include frameshifts integrated into repetitive GC-sequences and G-runs known to be mutagenic hot-spots, and base-pair substitutions inserted in or around the beta-lactamase active site. Frameshift mutations completely inactivated the enzyme only when located downstream of the active-site serine (Ser68). Previous (reporter gene based) assays with this system have detected reversion to ampicillin resistance by luminescence driven by induction of the tet-promotor controlled lacZ gene. In the present study, we describe the construction and evaluation of 19 additional potential tester strains. We also developed conditions for detecting reversions by pH shift using bromocresol purple and by directly detecting the enzymatic activity of beta-lactamase using nitrocefin. A 384-well format version of the pH shift MutaGen test was used to assay more than 20 chemicals. The responses in the assay were compared with responses for the same chemicals in the umu test and Ames fluctuation assays. The results indicate that the MutaGen test has high specificity for detecting specific mutations and, in some instances, better sensitivity than the other tests. Since the test is easy to conduct, sterile working conditions are not necessary, and the mutagenicity results are available either within one working day or overnight, the assay shows promise for the rapid screening of potentially genotoxic substances.
Environ Mol Mutagen 2005 Aug
PMID:Further development of the beta-lactamase MutaGen assay and evaluation by comparison with Ames fluctuation tests and the umu test. 1588 Jul 35


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