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PubMed1987

The movement of melanosomes in melanophore fragments obtained by laser microbeam irradiation.

The melanophores of the teleost Gymnocorymbus ternetzi are filled with pigment granules, melanosomes, which in response to appropriate treatments, can disperse throughout the cytoplasm or form an aggregate in the cell center. Melanophores with the dispersed pigment were irradiated by a laser microbeam, focused on the cell center by the microscope objective. If the average energy of the microbeam was 6-7 microJ, either the center of the melanophore was damaged and a single ring-shaped fragment was formed, or the cell was broken into several fragments of smaller size. The fragments retained their ability to move the pigment granules. In ring-shaped fragments, after adrenaline treatment, the melanosomes formed a ring-shaped aggregate moving away from both outer and inner (irradiation-produced) margins of the fragment. The smaller fragments treated with adrenaline moved the pigment to their centers. Both small and ring-shaped fragments could aggregate melanosomes as soon as 5 minutes after irradiation.

Animals↗
PubMed1976

Propagation in E. coli of bacteriophage lambda with integrated fragments of adenovirus 2 DNA.

Hybrid genomes of bacteriophage lambda with integrated fragments of adenovirus type 2 (Ad2) DNA have been constructed in vitro and propagated in E. coli. DNA from a derivative of bacteriophage lambdaplac5 (Rambach and Tiollais, 1974) was used as a vector. The two fragments of the vector DNA contain all the essential genes for the replication of the lambda DNA but are too short to be encapsidated. Insertion of DNA is therefore essential for plaque formation which constitutes a selection method for phages containing hybrid genomes. Fragments EcoRI-B and EcoRI-F of Ad2 DNA were purified, and separately ligated with the vector fragments. Clones of hybrid phage could readily be isolated. Two clones of hybrid phage containing fragment EcoRI-B inserted in opposite directions were used to study the transcription of adenovirus-specific sequences. Hybridization experiments showed that transcripts from both strands of fragment Ad2-Eco RI-B could be detected and that transcription probably was controlled by the "early" leftward and the "late" rightward promoters on the lambda genome. No polypeptides specified by the adenovirus fragment have so far been identified.

Adenoviridae↗
PubMedDate not supplied

A technique for integrating any DNA fragment into the chromosome of Escherichia coli.

We describe a technique that allows the insertion of any DNA fragment into the EcoRI-site-containing malPpa, the promoter of malPQ, one of the three maltose operons of Escherichia coli. DNA fragments were cloned into the unique EcoRI site of the pBR322-derived plasmid pOM40, which carries malPpa. In the next step these fragments were transposed into the chromosome by homologous recombination events occurring on both sides of malPp. Cells in which such insertion of the entire recombinant plasmid have occurred can be conveniently selected. Excision and curing of the vector plasmid could then occur spontaneously at a high frequency, leaving behind the inserted fragment that can be manipulated as any chromosomal marker. When the inserted fragment contains a properly positioned promoter, its promoting activity can be estimated by assaying amylomaltase, the product of malQ. When required, the inserted fragment can be easily transferred back onto pOM40. As examples of application we have transferred two different fragments into the chromosome of E. coli: one contained the ceaC-ceiC operon, which encodes colicin E3 and its immunity protein, and the other contained the lac promoter of E. coli.

Base Sequence↗
PubMed1992

Shotgun polymerase chain reaction: construction of clone libraries specific to a NotI fragment of flow-sorted human chromosome 22.

We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.

Base Sequence↗
PubMed1995

A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGF is a cell growth inhibitor.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is now recognized as a normal process in the regulation of insulin-like growth factor (IGF) activity, its major effect being to increase IGF bioavialability. In order to characterize the proteolytic fragments of IGFBP-3, we reproduced this proteolysis in vitro using plasmin which provokes cleavages that are similar to those induced in vivo by (unidentified) specific IGFBP-3 proteases. Two major peaks were purified by RP-HPLC. One contained a 16 kDa fragment and the other comprised two fragments of 22 and 25 kDa. Competitive binding experiments showed that the 16 kDa material had no affinity for IGFs. The 22-25 kDa fragments had considerably reduced affinity, particularly for IGF-I. In a chick embryo fibroblast assay where DNA synthesis was stimulated by IGF-I or insulin, the 22-25 kDa fragments weakly inhibited IGF-I-induced cell proliferation and had no effect on stimulation by insulin. The 16 kDa fragment unexpectedly proved to be a potent inhibitor of both IGF- and insulin-induced cell growth. This proteolytic fragment of IGFBP-3 therefore exhibits intrinsic inhibitory activity.

Animals↗
PubMed2006

Site-directed immobilisation of antibody fragments for detection of C-reactive protein.

C-reactive protein, CRP antibody Fab'-fragments have been attached on pre-cleaned gold slides and protein repellent polymers have been used to block the remaining free space in between the antibody fragments. At optimal conditions the antibody fragments are site-directly immobilised on the surface and non-specific binding is reduced. The amount of Fab'-fragments in the polymer host monolayer has been optimised for various buffers. Binding of CRP to Fab'-fragment/polymer layers produced in phosphate buffered saline decreased with NaCl salt concentration. In a 1M NaCl phosphate buffer, the antibodies seem to be randomly oriented on the surface with a similar response to CRP as that of an antibody F(ab)(2)-fragment layer. In a 150 mM NaCl phosphate buffer, on the other hand, the fragments seem to be site-directly oriented and the response to CRP was fivefold. The highest response to CRP was obtained to a layer with a Fab'-fragment concentration of 60 microg/ml. CRP could be detected in a concentration range of 1 ng/ml to 50 microg/ml from a standard solution in phosphate buffer and in a range of 4 ng/ml to 50 microg/ml from serum/PBS. CRP was, moreover, successfully detected in patient samples with good reproducibility. The layer would thus be sensitive enough to analyse the CRP concentration in human serum for predicting cardiovascular disease.

Adsorption↗
PubMed2006

Sequential resolution of fragmented visual percepts: experimental investigation of a subject's perceptual experience after a right medial temporal stroke.

This report concerns the fragmented visual percepts in a woman, TR, following a right entorhinal-perirhinal infarct. In a previous report, Weddell [Weddell, R. A. (2005). A visual disorder producing highly selective deletion of recurring letters. Cortex, 41, 471-485] linked TR's highly selective tendency to delete recurrent letters with her fragmented percepts. The conflation of same-identity form elements was attributed to anterior extrastriate damage, which reduced the amount of information sustainable in fully resolved visual percepts, and the present experimental investigation of her subjective account of segment formation and resolution completes the story. She said that complex objects and long words first appeared as blurred regions, which sometimes included form elements. It is argued that figure-centred attentional mechanisms subdivided this blurred region into up to 3-4 parts. String length, lexical status (word vs. nonword), and background colour and/or luminance determined fragment length. Two rules described the fragment resolution sequence: largest segments usually resolved first, left-to-right resolution accounting for a few sequences. This resolution sequence occurred when stimuli were exposed too briefly for saccadic exploration, implicating endogenous attentional shifts. Experiment 4 confirmed TR's assertion that spatial, orthographic, and phonological information were stored during the fragment resolution process. Moreover, TR exerted considerable voluntarily control over the fragment resolution sequence and some influence over fragment length. Finally, these findings were interpreted in terms of an extended version of a neural network model of vision largely derived from nonhuman primate studies.

Adult↗
PubMed2004

Fragmentation pathways of N(G)-methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS.

Protein methylation at arginine residues is a prevalent posttranslational modification in eukaryotic cells that has been implicated in processes from RNA-binding and transporting to protein sorting and transcription activation. Three main forms of methylarginine have been identified: N(G)-monomethylarginine (MMA), asymmetric N(G),N(G)-dimethylarginine (aDMA), and symmetric N(G),N'(G)-dimethylarginine (sDMA). To investigate gas-phase fragmentations and characteristic ions arising from methylated and unmodified arginine residues in detail, we subjected peptides containing these residues to electrospray triple-quadrupole tandem mass spectrometry. A variety of low mass ions including (methylated) ammonium, carbodiimidium, and guanidinium ions were observed. Fragment ions resulting from the loss of the corresponding neutral fragments (amines, carbodiimide, and guanidine) from intact molecular ions as well as from N- and C-terminal fragment ions were also identified. Furthermore, the peptides containing either methylated or unmodified arginines gave rise to abundant fragment ions at m/z 70, 112, and 115, for which cyclic ion structures are proposed. Electrospray ionization tandem mass spectra revealed that dimethylammonium (m/z 46) is a specific marker ion for aDMA. A precursor ion scanning method utilizing this fragment ion was developed, which allowed sensitive and specific detection of aDMA-containing peptides even in the presence of a five-fold excess of phosphorylase B digest. Interestingly, regular matrix-assisted laser desorption/ionization mass spectra recorded from aDMA- or sDMA-containing peptides showed metastable fragment ions resulting from cleavages of the arginine side chains. The neutral losses of mono- and dimethylamines permit the differentiation between aDMA and sDMA.

Arginine↗
PubMed2004

Establishing low-energy sequential decomposition pathways of leucine enkephalin and its N- and C-terminus fragments using multiple-resonance CID in quadrupolar ion guide.

The simultaneous resonant low-energy excitation of leucine enkephalin and its fragment ions was demonstrated in a collision cell of the multipole-quadrupole time-of-flight instrument. Using low-amplitude multiple-resonance excitation CID, we were able to show the exclusive sequential fragmentation of N- and C-terminus fragments all the way to the final fragments--immonium ions of phenylalanine or tyrosine. In this CID mode the single-channel dissociation of each new generation of fragments followed the lowest energy decomposition pathways observable on the time scale of our experiment. Up to six generations of sequential dissociation were carried out in multiple-resonance CID experiments. The direct qualitative comparison of fragmentation of axial-acceleration versus resonant (radial) CID was performed in the same instrument. In both activation methods, fragmentation patterns suggested complex decomposition mechanisms attributable to dynamic competition between sequential and parallel dissociation channels.

Enkephalins↗
PubMed2005

Influence of the labeling group on ionization and fragmentation of carbohydrates in mass spectrometry.

The ionization and fragmentation behaviors of carbohydrate derivatives prepared by reaction with 2-aminobenzamide (AB), 1-phenyl-3-methyl-5-pyrazolone (PMP), and phenylhydrazine (PHN) were compared under identical mass spectrometric conditions. It has been shown that the intensities of signals in MS spectra depend on the kind of saccharides investigated and reducing end labels used. PMP sialyllactose, when ionized by ESI/MALDI, produced a mixture of [M + H]+, [M + Na]+, [M - H + 2Na]+ ions in the positive mode and [M - H]-, [M + Na - 2H]- ions in the negative mode. The AB and PHN derivatives formed abundant [M + H]+ and [M - H]- ions in ESI, and by matrix-assisted laser desorption/ionization (MALDI) produced abundant [M + Na]+ ions. PMP- and reduced AB-sialyllactose produced only Y-type fragment ions under both MS/MS sources. In the electrospray ionization (ESI)-MS/MS spectrum of PHN-sialyllactose, abundant ions corresponded to B, Z cleavages and in its MALDI-MS/MS spectrum, the abundant ions were consistent with Y glycosidic cleavages with the concurrence of B, C, and cross-ring fragment ions. In the MALDI-MS spectra of oligosaccharides acquired immediately after derivatization, it was possible to detect only PHN derivatives. After purification, spectra of all three types of derivatives showed high signal-to-noise ratios with the most abundant ions observed for AB reduced saccharides. [M + Na]+ ions were the dominant products and their fragmentation patterns were influenced by the type of the labeling and the kind of oligosaccharide considered. In the MALDI-PSD and -MS/MS spectra of AB-derivatized glycans, higher m/z fragment ions corresponded to B and Y cleavages and the loss of bisecting GlcNAc appeared as a weak signal or was not detected at all. Fragmentation patterns observed in the spectra of hybrid/complex PHN and PMP glycans were more comparable-higher m/z fragments corresponded to B and C glycosidic cleavages. For PHN glycans, the abundance of ions resulting from the loss of bisecting GlcNAc depended on the number of residues linked to the 6-positioned mannose. Also, PHN and PMP derivatives produced cross-ring cleavages with abundances higher than observed in the spectra of AB derivatized oligosaccharides. For high-mannose glycans, the most informative cleavages were provided by AB and PHN type of labeling. Here, PMP produced dominant Y-cleavages from the chitobiose while other ions produced weak signals.

Antipyrine↗
PubMed2005

Biochemical screening of stable dinucleosomes using DNA fragments from a dinucleosome DNA library.

The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.

Animals↗
PubMed2004

c-Jun N-terminal kinase and, to a lesser extent, p38 mitogen-activated protein kinase regulate inducible nitric oxide synthase expression in hyaluronan fragments-stimulated BV-2 microglia.

Lower molecular weight of hyaluronan (HA) fragments are capable of activating macrophages to express a number of inflammatory mediators through the interaction with the HA receptor CD44. Recent evidence has demonstrated that concomitant induction of CD44 and HA synthase 2 (HAS-2) mRNA in microglia of the ischemic brain. However, the influence of HA fragments on the activation of microglia is poorly understood. In this study, we demonstrated that HA fragments induced inducible NO synthase (iNOS) expression in BV-2 microglia in a dose-dependent manner and was synergized with interferon-gamma (IFN-gamma). Moreover, HA fragments could induce the activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/ERK2), and c-Jun N-terminal kinase (JNK) in a time and dose-dependent fashion. The HA fragments-induced iNOS expression was suppressed by the selective inhibitors of JNK and, to a lesser extent, p38 MAPK. These results suggest that the induction of iNOS by HA fragments is significantly dependent on JNK than on p38 MAPK signaling pathways and support the hypothesis that HA fragments may be an important regulator in the activation of microglia at sites of ischemic brain.

Animals↗
PubMed2004

Fibronectin fragments cause release and degradation of collagen-binding molecules from equine explant cultures.

OBJECTIVE: Previous experiments have shown that addition of fragmented fibronectin can induce cartilage chondrolysis. In this study we investigated the fate of the collagen- and cell-binding molecules Cartilage oligomeric matrix protein (COMP) and chondroadherin. DESIGN: Equine articular cartilage explants were stimulated with the C-terminal and the N-terminal heparin-binding fragments of fibronectin respectively, and the conditioned media were analysed by both quantitative (ELISA) and qualitative (mass spectrometry, Western blots) methods. RESULTS: Both COMP and chondroadherin were released in a dose-dependent manner upon stimulation with the Hep II (C-terminal heparin-binding) fragment of fibronectin. The kinetics of release for the two components differed. Moreover, COMP was degraded while no fragments of chondroadherin could be detected. Stimulation with Hep II also induced production of nitric oxide in a dose-dependent manner. We compared effects of the Hep II fragment with that of Hep I (the N-terminal heparin-binding fragment of fibronectin) and found that while Hep I did indeed elicit release of COMP and chondroadherin, the response was less potent, and production of nitric oxide was negligible. The responses to both fragments were elicited within 24h. CONCLUSIONS: We suggest that the events described here may be early, critical stages in cartilage destruction preceding collagen destruction.

Animals↗
PubMed2004

Sub-fragments of the envelope gene are highly protective against the Japanese encephalitis virus lethal infection in DNA priming--protein boosting immunization strategies.

The envelope (E) gene of Japanese encephalitis virus (JEV) plays a major protective role against JEV infection. In order to locate the part of E gene that is responsible for this protection, an N-terminal fragment EA (nucleotide number 933-1877 bp of JEV genome) and a C-terminal fragment EB (nucleotide number 1851-2330 bp of JEV genome) from E gene were prepared. Both of these fragments were used in the form of recombinant proteins (rEA and rEB) and plasmid DNA (pEA, pM15EA and pEB) for immunizations. Recombinant EA protein (rEA) was previously found to be non-protective because it was expressed in an insoluble form. Plasmid EA (pEA) was also found to be non-protective unless it is preceded by a 15 mer signal peptide derived from the very C-terminal of the membrane gene (M) of JEV to form pM15EA plasmid indicating the importance of the signal peptide in the expression of EA immunogenicity. Although pM15EA and pEB are both immunogenic and protective against JEV lethal infection, the protection by both fragments however is not optimal. Even when pM15EA and pEB were used together for immunization, maximum protection as those induced by control vaccine was not achieved. However, if individual fragments (EA or EB) were used in a DNA priming-protein boosting or protein priming-DNA boosting strategy, high levels of protection were achieved by both fragments. This was especially true for EA fragment where the level of protection against JEV lethal infection was equal to that induced by commercially available vaccine alone. The protection correlated very well with the neutralizing antibody titers and the T helper cell involved in this process in mainly the Th1 type.

Animals↗
PubMed1999

Elevated maternal urine level of beta-core fragment of human chorionic gonadotropin versus serum triple test in the second-trimester detection of down syndrome.

OBJECTIVE: This study was undertaken to compare the Down syndrome screening efficiency of elevated maternal urine level of the beta-core fragment of human chorionic gonadotropin with that of the traditional serum triple test. STUDY DESIGN: Urinary beta-core fragment and serum analyte levels were measured prospectively in women with singleton pregnancies who were undergoing second-trimester genetic amniocentesis. Urinary analyte levels were measured within a week of specimen collection. In some cases only alpha-fetoprotein was measured initially and human chorionic gonadotropin and unconjugated estriol levels were subsequently determined from the stored serum specimens. The Down syndrome screening efficiency of urinary concentration of beta-core fragment plus maternal age was compared with that of the traditional triple test. Receiver operating characteristic curves were generated for each algorithm and the areas under the curves were compared to determine which algorithm was superior. RESULTS: There were a total of 926 study patients, of whom 21 (2.3%) carried fetuses with Down syndrome. The mean (+/-SD) gestations at amniocentesis were 16.6 +/- 1.5 weeks for the fetuses without Down syndrome and 17.7 +/- 2.3 for the fetuses with Down syndrome. A total of 539 women (4 of whom carried fetuses with Down syndrome) had serum alpha-fetoprotein alone measured initially. Urinary concentration of beta-core fragment had a 61.9% detection rate with a 4.9% false-positive rate for Down syndrome, whereas the values for the triple screen were 57. 1% and 11.2%, respectively. The areas under the receiver-operating characteristic curves were 0.8744 for elevated urinary beta-core fragment level and 0.7504 for the triple screen (P =.1116). When the false-positive rate was fixed at an ideal threshold value (</=5%) the urine test was superior (area under the curve, 0.0212 vs 0.0133, P <.05). Similarly, when we considered only cases in which the complete triple screen was performed prospectively (17 fetuses with Down syndrome and 431 fetuses without Down syndrome), the urine test was significantly better (area under the curve, 0.873 vs 0.624, P =.012). CONCLUSION: In this first reported direct comparison we consistently observed higher sensitivity values for screening with urinary levels of beta-core fragment than for serum triple screen, suggesting an equivalent or superior Down syndrome screening performance for the urinary analyte. It is important that freezing and prolonged urine storage before testing be avoided. The reduced cost (single- versus triple-analyte testing) and excellent screening performance support large-scale testing and evaluation of maternal urinary beta-core fragment measurement as an alternative to the traditional serum triple test.

Adult↗
PubMed2005

Fragmentation of the Golgi apparatus induced by the overexpression of wild-type and mutant human tau forms in neurons.

Tau is a microtubule-associated protein enriched in the axonal compartment. In several neurodegenerative diseases including Alzheimer's disease, hyperphosphorylated tau accumulates in the somatodendritic compartment, self-aggregates, and forms neurofibrillary tangles. A fragmentation of the neuronal Golgi apparatus (GA) was also observed in Alzheimer's disease. In the present study, we examined the effect of overexpressing human tau on the organization of the neuronal GA in rat hippocampal cultures and in JNPL3 mice expressing tau mutant P301L. GA fragmentation was noted in a significantly higher percentage of hippocampal neurons overexpressing wild-type human tau than in control neurons over-expressing green fluorescent protein (GFP) alone. Most importantly, in neurons overexpressing mutant forms of human tau (P301L, V337M, or R406W), the percentage of neurons with a fragmented GA was 10% higher than that of neurons overexpressing wild-type human tau. In JNPL3 mice, a significantly higher percentage of motor neurons presented a fragmented GA compared to control mice. Interestingly, fragmentation of the GA was more frequent in neurons containing an accumulation and aggregation of hyperphosphorylated tau in the cell body than in neurons without these features. In both primary hippocampal neurons and JNPL3 mice, the tau-induced GA fragmentation was not caused by apoptosis. The pre-sent results implicate tau in GA fragmentation and show that this event occurs before the formation of neurofibrillary tangles.

Animals↗
PubMed2001

The binding of cyanide to cytochrome d in intact cells, spheroplasts, membrane fragments and solubilized enzyme from Salmonella typhimurium.

This investigation focused on the kinetics of cyanide binding to oxidized and reduced cytochrome d in Salmonella typhimurium intact cells, spheroplasts, membrane fragments and solubilized enzyme, and on the effect of pH on this binding. Cyanide bound to the oxidized form of cytochrome d under all experimental conditions, inducing a trough at 649 nm in the oxidized-cyanide-minus-oxidized difference absorption spectra. V(max) of cyanide binding to oxidized cytochrome d at pH 7.0 was 14.0+/-2.0 pmol/min/mg protein (prot.) in intact cells, 37.0+/-3.5 pmol/min/mg prot. in spheroplasts, 125.0+/-6.0 pmol/min/mg prot. in membrane fragments, and 538.0+/-8.5 pmol/min/mg prot. in solubilized cytochrome d. The pseudo-first order rate constants were 0.004 s(-1) for intact cells, 0.005 s(-1) for spheroplasts, 0.007 s(-1) for membrane fragments and 0.025 s(-1) for the solubilized enzyme. The V(max) value was highest at pH 7.0 for intact cells and solubilized cytochrome d and at pH 8.0 for both spheroplasts and membrane fragments. The K(s) of binding at pH 7.0 was around 4 mM in intact cells, spheroplasts and membrane fragments, but was 10.5 mM in solubilized cytochrome d. This difference between the K(s) values suggested a change in conformation, upon solubilization, leading to a decrease in the affinity of cyanide for the solubilized enzyme. The K(s) value was nearly the same at all pH investigated (pH 5-10). Cyanide was found to also bind to the reduced form of cytochrome d in membrane fragments (K(s)=18+/-3 mM, V(max)=377+/-28 pmol/min/mg prot. at pH 7) and the solubilized enzyme (K(s)=18+/-1.2 mM, V(max)=649+/-45 pmol/min/mg prot. at pH 7) with a lower affinity of cyanide for the reduced cytochrome d than for the oxidized enzyme. Pseudo-first order rate constants were 0.025 s(-1) and 0.042 s(-1) respectively for membrane fragments and solubilized enzyme. The value of V(max) for cyanide binding to the reduced cytochrome d, whether membrane-bound or solubilized, increased slightly with pH (for pH 6-10) while the K(s) value dropped significantly with increasing pH. The pH dependence observed here might be interpretable as a possible role for conformational transition associated with energy transduction. Finally, this investigation pointed to the influence of the microenvironment of a protein within the cell on its reactivity.

Cyanides↗
PubMed1999

Adhesion of human endometrial fragments to peritoneum in vitro.

OBJECTIVE: To evaluate the adhesion of endometrial fragments obtained during the proliferative phase of the menstrual cycle to fresh human peritoneum obtained during abdominal surgery. DESIGN: A prospective, descriptive, morphologic and cell biologic study. SETTING: Tertiary care university medical center. PATIENT(S): Six female volunteers. INTERVENTION(S): After endometrial biopsies performed during diagnostic laparoscopy, endometrial fragments were generated by enzymatic digestion and mechanical separation. Peritoneum was obtained during abdominal operations for benign indications. MAIN OUTCOME MEASURE(S): Adhesion of endometrial fragments was studied by histologic examination and scanning and transmission electron microscopy. RESULT(S): After incubation, the mesothelium was intact in some areas, whereas in other areas mesothelial cells were damaged or absent. Adhesion of endometrial fragments was observed only at locations where the basement membrane was exposed. In areas largely denuded of mesothelial cells, endometrial fragments spread over the basement membrane to form monolayers. CONCLUSION(S): Human peritoneum is suitable for studying the adhesion of endometrial fragments. Intact mesothelium prevents the adhesion of endometrial fragments, suggesting that trauma to the mesothelial lining is a prerequisite for endometrial cell adhesion.

Basement Membrane↗