Degree Of Cleavage Research Articles

Fattening and slaughter qualities of lambs under conditions of use in their diets of feeds with different degrees of cleavage in the rumen of protein

Materials of scientific and experimental studies are indicated, which indicate certain differences in the formation of productive qualities of young Askani sheep of fine-wooled breed during intensive fattening when consuming rations consisting of feed where protein had different degrees of cleavage in the rumen. It was found that when using for feeding lambs II experimental group rations similar in amount of crude protein, but not the same in the level of digestibility of protein fractions (31% of undigested protein versus 23% in control of total concentration) increased by 5.7% of their mercury weight at the age of 6.5 months, when the animals reached slaughter condition. This difference is primarily due to the better biotransformation of feed nutrients (7.4 ECO / kg growth versus 8.0 ECO / kg control) and high growth rate, which was 12.9% higher in young sheep of experimental group II (201 g / goal / day against 178 g / goal / day in control).
 When correcting the consumption of protein fractions digestible in the rumen, the carcass quality was improved in experimental lambs, namely by 5.7% of carcass weight (20.3 kg vs. 19.2 kg in the control) and 0.6 abs. % Of carcass yield (47, 3% vs. 46.7% in control).
 Morphological evaluation of animal carcasses showed that the optimization of protein norms by the degree of its cleavage in the rumen contributed to an increase in young sheep of the II experimental group by 1.7 abs. % (74.4% vs. 72.7%) of muscle tissue yield due to decreased bone and tendon mass. In view of this, the sheep of this group was larger by 0.25 units. meat content ratio (2.91 units vs. 2.66 units in the control). , 44 kg vs. 9.52 kg in control) while improving its biological and nutritional value.

Experiment and Smooth Particle Hydrodynamics Simulation for the Wear Characteristics of Single Diamond Grit Scratching on Sapphire

Abrasive single crystal diamond (SCD) grit is widely used in the machining process of sapphire. The wear of SCD grit has a significant influence on the surface quality of sapphire. In this paper, smooth particle hydrodynamics (SPH) method is employed to reveal the wear mechanism of SCD grit with Steinberg constitutive equation and Grüneisen state equation. The wear morphology, wear volume and scratching forces are measured and analyzed by combination of SPH simulations and experiments. The results show that the scratching forces fluctuate in a certain range and decrease with the increasing of workpiece material removal volume. Different degrees of cleavage and fracture appear in the front and rear of SCD grit. The shear stress and extrusion stress are the main stresses of SCD grit during the scratching process. The wear progress and wear form are mainly determined by the stress state. Different stress state leads to different wear progress of the SCD grit. The SPH method is able to reflect and illustrate the wear characteristics of SCD grit scratching on sapphire.

The Sequence Preference of Gamma-Radiation-Induced Damage in End-Labeled DNA after Heat Treatment.

In this work, we examined the DNA sequence preference of gamma-radiation-induced DNA damage in purified DNA sequences after heat treatment. DNA was fluorescently end-labeled and gamma-radiation-induced DNA cleavage was examined using capillary electrophoresis with laser-induced fluorescence detection. Our findings provide evidence that gamma-radiation-induced DNA damage to end-labeled DNA is nonrandom and has a sequence preference. The degree of cleavage was quantified at each nucleotide, and we observed that preferential cleavage occurred at C nucleotides with lesser cleavage at G nucleotides, while being very low at T nucleotides. The differences in percentage cleavage at individual nucleotides ranged up to sixfold. The DNA sequences surrounding the most intense radiation-induced DNA cleavage sites were examined and a consensus sequence 5'-AGGC*C (where C* is the cleavage site) was found. The highest intensity gamma-radiation-induced DNA cleavage sites were found at the dinucleotides, 5'-GG*, 5'-GC*, 5'-C*C and 5'-G*G and at the trinucleotides, 5'-GG*C, 5'-TC*A, 5'-GG*G and 5'-GC*C. These findings have implications for our understanding of ionizing radiation-induced DNA damage.

The Most Recent, Catalytically Fit HDV Ribozyme Exhibits Minimal Global and Small-Scale Conformational Change upon Cleavage

Historically, all small catalytic RNAs have been shown to undergo global conformational changes upon phosphodiester cleavage. However, the most recent of numerous hepatitis delta virus (HDV) ribozyme crystal structures has challenged this trend, as this crystal structure suggests that the precursor structure is already product-like in conformation. To further investigate this unusual observation, we have extensively characterized the solution behavior of several three-stranded versions of the HDV ribozyme from the recent crystal structure. Fluorescence gel shift assays show that varying lengths of the 5'overhang sequence adjacent to the active site result in the same degree of cleavage, whereas noncleavable substrates exhibit significantly more heterogeneity. Complementary steady state and time-resolved FRET assays demonstrated that the length of the 5'overhang sequence adjacent to the cleavage site affects the rates of conformational change upon substrate binding and cleavage. Molecular dynamics (MD) simulations were also performed to gain insight into the atomic behavior and catalytic relevance of the HDV ribozyme from the Chen et al crystal structure. These simulations suggested that the dU-1dG1dG2 motif used in the crystal does not result in a catalytically fit ribozyme compared to an all-ribose construct. Furthermore, altered active site conditions also result in lowered catalytic fitness. These simulations suggested that catalytic fitness is greatly disrupted by deprotonation of C75, supporting the hypothesis of the role of C75 as a general acid. Simulations also showed that the magnesium ion resolved near the scissile phosphate results in favorable catalytic geometry compared to simulations neutralized with sodium. Our experimental results demonstrate that, despite previously published results, all forms of the HDV ribozyme undergo significant global conformational changes upon self-cleavage, and our simulations show that C75 is poised to act as a general acid during cleavage.

Cytotoxic Rhodium(III) Polypyridyl Complexes Containing the Tris(pyrazolyl)methane Coligand: Synthesis, DNA Binding Properties and Structure–Activity Relationships

AbstractThe RhIII complexes of the type [RhCl(pp)(tpm)]2+ [pp = bpy, bpm, phen, tap, dpq, dppz] 4–9 have been prepared by stepwise treatment of RhCl3·3H2O or mer,cis‐[RhCl3(DMSO‐κS)2(DMSO‐κO)] with the appropriate polypyridyl ligand (pp) followed by the tripodal ligand tris(pyrazolyl)methane (tpm). Intermediates of the type [RhCl3(CH3OH)(pp)] 1–3 with pp = bpy, phen, dpq were also characterized but exhibit either low (3) or no (1, 2) cytotoxicity. X‐ray structural analyses of [RhCl(bpy)(tpm)][PF6]2 4 and [RhCl(phen)(tpm)][PF6]2 6 were performed, and the interaction of complexes 4–9 with DNA was investigated by CD and UV/Vis spectroscopy and by gel electrophoresis. CD and viscosity studies confirm strong intercalation of dppz complex 9 into DNA. Complexes 8 and particularly 9 (IC50 = 0.43, 0.37 μM) are potent cytotoxic agents towards the human cancer cell lines MCF‐7 and HT‐29, whereas respectively little (complex 6) or no activity (complexes 4, 5, 7) is observed for the other members of the series. Our findings indicate that the cytotoxicity is dependent on the hydrophobicity of both the polypyridyl and the facial coligand in these and other half‐sandwich RhIII complexes. Irradiation of bpy compound 4 in the presence of plasmid pBR322 for 30 min at 311 nm at a molar ratio of r = 0.1 leads to total conversion of the supercoiled form into the nicked version. Although dppz complex 9 also functions as a photonuclease under these conditions, the degree of cleavage is much lower. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Inhibition of the Proteolytic Activity of Pregnancy-associated Plasma Protein-A by Targeting Substrate Exosite Binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.

Cleavage of the Notch Ligand Delta‐1 in the Regulation of T Cell Development

Signaling through the Notch ligand, Delta1 (DL1) facilitates normal thymocyte development. DL1 is presented to thymocytes by thymic epithelial cells (TECs) and can be proteolytically cleaved by ADAM proteases. However, the roles of DL1 cleavage products remain controversial. Mice lacking ADAM17 in TECs display blocks in T cell development, leading to speculation that Notch signaling between TECs and thymocytes is aberrant in these animals. However, this has yet to be experimentally tested. The objective of the current research is to determine whether ADAM17‐mediated cleavage of DL1 in TECs facilitates successful Notch signaling during thymocyte development. The OP9‐DL1 co‐culture system allows for the generation of mature T cells in vitro. Western blotting of OP9‐DL1 lysates with an intracellular Ab to DL1 showed steady state cleavage of DL1 and that the degree of cleavage is modulated by activators and inhibitors of ADAM proteases. Furthermore, co‐culture of thymocytes on OP9‐DL1 cells leads to an upregulation in DL1 cleavage. Non‐cleavable DL1 mutants and ADAM17 shRNA will be used to study the function of DL1 cleavage on thymocyte development in the OP9‐DL1 system. These studies will lend insight into the role of DL1 cleavage in the regulation of Notch signaling. Moreover, these data will provide evidence for whether processing of Delta1 is required for proper intrathymic cellular communication.

Characterization of cis-Autoproteolysis of Polycystin-1, the Product of Human Polycystic Kidney Disease 1 Gene

Polycystin-1 (PC1), the PKD1 gene product, plays a critical role in renal tubule diameter control and disruption of its function causes cyst formation in human autosomal dominant polycystic kidney disease. Recent evidence shows that PC1 undergoes cleavage at the juxtamembrane G protein-coupled receptor proteolytic site (GPS), a process likely to be essential for its biological activity. Here we further characterized the proteolytic cleavage of PC1 at the GPS domain. We determined the actual cleavage site to be between leucine and threonine of the tripeptide HLT(3049) of human PC1. Cleavage occurs in the early intracellular secretory pathway and requires initial N-glycan attachment but not its subsequent trimming. We provide evidence that the cleavage occurs via a cis-autoproteolytic mechanism involving an ester intermediate as shown for Ntn hydrolases and EMR2.

A Substrate Specificity-determining Unit of Three Lin12-Notch Repeat Modules Is Formed in Trans within the Pappalysin-1 Dimer and Requires a Sequence Stretch C-terminal to the Third Module

Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP-A cleaves IGFBP-4 and IGFBP-5, whereas PAPP-A2 cleaves only IGFBP-5. The pappalysins contain three Lin12-Notch repeat (LNR1-3) modules, previously considered unique to the Notch receptor family in which they function to regulate receptor cleavage. In contrast to the Notch receptor where three LNR modules are tandemly arranged, LNR3 is separated by more than 1000 residues from LNR1-2 in the pappalysin sequence. Each of the three LNR modules of PAPP-A is required for proteolysis of IGFBP-4, but not IGFBP-5. However, we here find that a C-terminal truncated variant of PAPP-A, which lacks LNR3 and therefore activity against IGFBP-4, cleaves IGFBP-4 when co-expressed with a PAPP-A variant, which is mutated in the active site. This suggests that LNR3 from the inactive subunit interacts in trans with LNR1-2 of the truncated PAPP-A subunit to form a functional trimeric LNR unit. We also show that formation of such a functional LNR unit depends on dimerization, as dissociation of a mutated non-covalent PAPP-A dimer results in reduced activity against IGFBP-4, but not IGFBP-5. Using PAPP-A/PAPP-A2 chimeras, we demonstrate that PAPP-A2 LNR1-2, but not LNR3, are functionally conserved with respect to IGFBP proteolysis. Additionally, we find that a sequence stretch C-terminal to LNR3 and single residues (Asp1521, Arg1529, and Asp1530) within this are required for LNR functionality.

Proteolytic processing of a human salivary proline-rich protein precursor by proprotein convertases.

Salivary proline-rich proteins (PRPs) are synthesized as precursors that are cleaved before secretion giving rise to glycosylated PRPs which have lubricating function and basic PRPs which are potent precipitators of dietary tannins. The putative cleavage sites in the precursors for basic and glycosylated PRPs all conform to the sequence RSXR downward arrowS (X can be A, S or P) in agreement with the recognition sequence (RXXR downward arrow) for various proprotein convertases. PRB4S, a proprotein giving rise to a basic PRP (IB-5) as well as a glycosylated PRP (II-1) was synthesized by in vitro transcription-translation. It was cleaved by furin at RSAR downward arrowS(173-178) giving rise to two proteins II-1 and IB-5. Similarly another precursor with the sequence RSAR downward arrowS(173-178) was also cleaved by furin. This together with previous results show that in vitro furin can cleave all RSXR downward arrowS sequences in the proproteins that give rise to glycosylated and basic PRPs. To demonstrate cellular cleavage, a human submandibular cell line (HSG) was transfected with a vector encoding PRB4S. This resulted in secretion of II-1 and IB-5. The degree of cleavage was enhanced by coexpressing furin and PRB4S. No cleavage occurred if the cells expressed a mutant PRB4S, R177Q, where the furin cleavage site had been destroyed. Cleavage was also inhibited if a furin inhibitor was coexpressed with PRB4S. Incubating the cells at 20 degrees C which blocks exit of proteins from the trans-Golgi network demonstrated that cleavage occurs before exit of the proteins from this network. These results show that furin may be responsible for in vivo cleavage of PRP precursors. Transfecting furin-deficient RPE.40 cells with a vector encoding PRB4S also led to secretion of II-1 and IB-5 showing that convertases other than furin can also cleave PRB4S in tissue culture.

Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor.

To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I-labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N-linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N-linked glycan. Elimination by mutagenesis of the most strategic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavage. Removal of the other N-linked glycans had no additive effect. A much greater degree of cleavage ( approximately 50%) was evident in a chimeric LH-TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317-367) was replaced with the corresponding domain of the TSHR (residues 363-418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR-NET317-319 replaced with TSHR-GQE367-369, or by substitution of the same three amino-acid residues with AAA (LHR-NET317-319AAA). In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) amino-acid specificity in a region important for TSHR cleavage (GQE367-369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.

Experimental evaluation of the ribonuclease protection assay method for the assessment of genetic heterogeneity in populations of RNA viruses.

The ribonuclease (RNase) protection assay (RPA) was evaluated as a method to estimate genetic distances among sequence variants of RNA viruses. The patterns of fragments generated, under different RPA conditions, by three sets of RNA sequence variants of known nucleotide sequence, were analyzed. Both the effectiveness of cleavage (i.e. the probability of cleavage in a certain heteroduplex) and its degree (i.e. in all the molecules in the assay or in a part of them) varied largely according to the nature of the mismatch. Probability and degree of cleavage were also dependent on distant sequence context effects. No correlation could be established between context and cleavage, so that the pattern of fragments in RPA cannot be unequivocally predicted from sequence information. Accordingly, nucleotide sequence differences between two sequence variants cannot be directly derived from RPA data. For all three sequence sets linear relationships were found between the number of non-shared fragments in the RPAs of two variants and their nucleotide sequence differences. Nevertheless, both linearity and the linear regression parameters varied largely according to the sequence set and according to RPA conditions, in a non-predictable way. Thus, under experimental conditions, RPA may not be as appropriate a method to estimate genetic distances between RNA sequences as simulation under an ideal model suggested. Possible ways to diminish the gap between the ideal model and the experimental procedure are proposed.

Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.

Endonuclease Activities Associated with High Molecular Weight and Internucleosomal DNA Fragmentation in Apoptosis

We have used pulsed-field gel electrophoresis and electron microscopy to correlate stages of DNA fragmentation with alterations of nuclear structure during apoptosis. DNA fragmentation occurs in two stages. The first is initiated by a previously undescribed endonucleolytic activity that cleaves DNA into 50- to 300kb fragments. Electron microscopy showed that this degree of cleavage was sufficient to cause the chromatin to undergo condensation. The second stage of fragmentation is catalyzed by the previously described calcium-magnesium endonuclease. The enzyme activity responsible for the initial fragmentation of DNA was found to be distinct from that causing subsequent internucleosomal DNA cleavage based upon its cation requirements, independence of proteolysis and lack of inhibition by zinc. Both activities were found to preexist in nuclei from thymocytes, liver, HL60, and IL2-dependent CTLL cells. Thus, in apoptosis DNA degradation involves two distinct endonucleolytic activities, with only the first activity being essential for cell death.

In vitro digestion of dystrophin by calcium-dependent proteases, calpains I and II

Dystrophin is a cytoskeletal protein which is thought to play an important role in membrane physiology since its absence (due to gene deficiency) leads to the symptoms of Duchenne muscular dystrophy (DMD). Some disruption in the regulation of intracellular free Ca 2+ levels could lead to DMD-like symptoms. In this study, calpains, which are very active calcium-dependent proteases, were examined for their capacity to hydrolyse dystrophin in vitro. The results show that calpains are able to split dystrophin and produce breakdown products of different sizes (the degree of cleavage being dependent on the incubation time with proteases). The time-course of protease degradation was examined by Western immunoblot using three polyclonal sera which were characterized as being specific to the central (residues 1173–1728) and two distal parts of the molecule ie specific to the N-terminal (residues 43– 760) or the C-terminal (residues 3357–3660) extremities of the dystrophin molecule. The cleavage patterns of dystrophin showed an accumulation of some major protease-resistant fragments of high relative molecular mass (250–370 kDa). These observations demonstrate that calpains digest dystrophin very rapidly when the calcium concentration is compatible with their activation. For instance, it is clear that calpains first give rise to large dystrophin products in which the C-terminal region is lacking. These observations suggest that dystrophin antibodies specific to the central domain of the molecule should be used to detect dystrophin for diagnostic purposes and before any conclusion as to the presence or absence of dystrophin can be deduced from results obtained using immunoanalyses of muscle biopsies.

Studies on the immunogenicity of chinese hamster ovary cell-derived recombinant gp120 (HIV-1IIIB)

Recombinant DNA-derived gp120 (HIV-1IIIB) expressed in chinese hamster ovary cells elicited specific humoral and cell-mediated immune responses in a variety of mammals. Antisera from immunized rabbits, sheep and goats recognized virus-derived gp120 and its precursor (gp160). Neutralizing antibodies were also elicited, but only in a few animals, and this may be related to the protein's susceptibility to cleavage through the neutralizing domain. However, in rabbits the degree of cleavage of gp120 had little or no effect on its antigenicity or immunogenicity. All antisera had limited cross-reactivity to envelope glycoproteins from a panel of HIV-1 isolates suggesting that immunodominant antibody epitopes are in variable regions of the recombinant gp120. Antigen-specific T-cell responses were detected in immunized macaques and were found to be stronger and more prolonged when gp120 was administered in Freund's adjuvant rather than alum.

Mass Spectra of N-Arylglycines and Substituted Aniline-N,N-Diacetic Acids

The electron impact mass spectra of eleven N-arylglycines and seven substituted aniline-N,N-diacetic acids were obtained under 70 eV and 14 eV ionization energies. The fragmentation patterns are similar to those of α-amino acids except for the degree of cleavage. The dehydration process of the diacetic acids may be considered as the characterestic process for this group of compounds.

Specificity towards oligomannoside and hybrid type glycans of the endo-?-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo-beta-N-acetylglucosaminidase (EC 3.2.1.96) named "Endo B", isolated from culture filtrates of the basidiomycete Sporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of the N-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43-49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)1(GlcNAc)3(Man)5(GlcNAc)2; (GlcNAc)3(Man)5(GlcNAc)2;(GlcNAc)3(Man)4(GlcNAc)2 were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)2(Man)5(GlcNAc)2 glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme. The oligomannoside- and the N-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule. Endo B represents a powerful tool for removing oligomannoside- and N-acetyllactosamine-type glycans from N-glycopeptides and N-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.

Steroid secretion by human oocyte--corona--cumulus complexes associated with conceptions following in vitro fertilization.

Seventy-one oocyte--corona--cumulus complexes (OCCCs) were obtained from eight women who conceived and delivered following in vitro fertilization (IVF) and eight women who were matched for the number of embryos transferred into the uterus but failed to conceive following the procedure. The steroid secretion of these OCCCs was assessed during a 24-hr culture period. Intermediate (N = 35) and mature (N = 36) complexes did not differ in steroid secretion, and progesterone was the major steroid secreted. OCCCs associated with a fertilized oocyte (N = 59) produced significantly (P less than 0.02) higher levels of progesterone than those associated with nonfertilized oocytes (N = 12) (515 +/- 53 vs 231 +/- 70 ng/OCCC/24 hr) but comparable levels of estradiol (9.17 +/- 1.14 vs 6.55 +/- 2.0 ng/OCCC/24 hr) and testosterone (0.34 +/- 0.09 vs 0.22 +/- 0.05 ng/OCCC/24 hr). Steroid production by OCCCs of oocytes which continued to cleave was not different from that of oocytes which did not cleave following fertilization, but the estradiol-to-testosterone ratio was significantly higher (31.4 +/- 3.5 vs 19.1 +/- 3.3; P less than 0.02) in the former group. The degree of cleavage was not related to the levels of steroid secretion. Cleaved oocytes associated with viable pregnancies (N = 28) were derived from OCCCs which secreted significantly less testosterone than those which did not result in a pregnancy following transfer (N = 26) (0.27 +/- 0.04 vs 0.41 +/- 0.04 ng/OCCC/24 hr).(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitivity to polar effects of reactions of homolytic substitution with cleavage of the C-Br bond and chain transfer constants in the radical telomerization of vinyl chloride with CBrCl3

1. The particular transfer constants were determined for radicals with a chain length from one to three monomer units in the radical telomerization of vinyl chloride with CBrCl3 at 55, 105, and 145°. 2. The polar properties of the reagents (estimated using the Bamford correlation equation) significantly influence the rate of homolytic reactions of substitution at the C-Br bond (in CBrCl3), and the sensitivity to polar effects is close to substitution at the C-Cl bond (in polychloromethanes) and substantially higher than in the case of cleavage of the C-H bond (in chloroform). 3. It was hypothesized that this is due to the higher degree of cleavage in the transition state of the weaker C-halogen bond and an increase in the probability of electron transfer or the role of ionic structures in the decrease in the activation energies of the corresponding substitution reactions.