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PubMed1992

Fragmentation of proteins in the 13- to 29-kDa mass range observed by 252Cf-plasma desorption mass spectrometry.

Results are presented on the observation of sequence-specific fragmentation of proteins in the 13- to 29-kDa mass range (ribonuclease A, papain, and proteinase K) by 252Cf-plasma desorption mass spectrometry. For these proteins, extensive N-terminal an, cn + 2, and dn fragment ions are observed with mass accuracies approaching 200 ppm for the most prominent fragment ions. The fragmentation pattern of these proteins shows the same fundamental behavior (arginine-directed, charge-remote fragmentation) that is observed in the fragmentation of small peptides, indicating that similar processes are occurring when large proteins and small peptides fragment in plasma desorption mass spectrometry.

Amino Acid Sequence↗
PubMed1994

Genetic variation detected by quantitative analysis of end-labeled genomic DNA fragments.

The continuing efforts to evaluate specific human populations for altered germinal mutation rates would profit from more efficient and more specific approaches than those of the past. To this end, we have explored the potential usefulness of two-dimensional electrophoresis of DNA fragments obtained from restriction-enzyme-digested genomic DNA. This permits the analysis, on a single preparation, of approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kb in the first dimension and from 0.3 to 2.0 kb in the second dimension. To enter into a genetic analysis, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately < or = 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variation that segregated within families according to Mendelian principles. We have established the feasibility of cloning a variant fragment from such gels and establishing its nucleotide sequence. This technology should be highly efficient in monitoring for mutations resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.

Base Sequence↗
PubMed1995

Properties of an N-terminal proteolytic fragment of apolipoprotein AI in solution and in reconstituted high density lipoproteins.

Limited proteolysis was used to study the domain structure and to produce a large N-terminal fragment of human apolipoprotein AI (apoAI). Digestion of reconstituted high density lipoprotein (rHDL) prepared with apoAI and dipalmitoyl phosphatidylcholine or palmitoyloleoyl phosphatidylcholine by chymotrypsin, trypsin, elastase, and subtilisin generated a major fragment of 22 kDa. Under milder conditions proteolysis of lipid-free apoAI produced a fragment of similar size. The fragments shared the same N terminus as intact apoAI, and the chymotryptic fragment had a molecular weight of 22,384 as determined by electrospray ionization mass spectrometry. Thus the fragment consists of the N-terminal 192 amino acid residues of apoAI, and the region around Tyr192 seems to be especially accessible to proteases. In aqueous solution the fragment, apoAI-(1-192), had an alpha-helix content similar to that of apoAI (approximately 52%) but existed only as monomers and dimers. ApoAI-(1-192) lysed dimyristoyl phosphatidylcholine liposomes slowly compared with apoAI but did form rHDL complexes with palmitoyloleoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine when prepared by the sodium cholate dialysis method. ApoAI-(1-192) rHDL exhibited sizes and size distributions distinct from apoAI rHDL but displayed similar stability against denaturation. The isolated apoAI-(1-192) rHDLs retained a high ability to activate lecithin-cholesterol acyltransferase, comparable with the most effective apoAI rHDL. The results suggest that the C-terminal domain of apoAI is crucial for self-association and initial lipid binding but is not involved in specific lecithin-cholesterol acyltransferase activation.

Amino Acid Sequence↗
PubMed1997

A 32-kDa proteolytic fragment of transcription factor Stat3 is capable of specific DNA binding.

Fragments of characteristic size retaining the ability of sequence-specific DNA binding were generated by partial proteolysis of transcription factor Stat3 with trypsin, chymotrypsin, or Staphylococcus V8 proteinase. The molecular masses of the smallest DNA-binding fragments were 75, 48, and 32 kDa after digestion with V8 proteinase, chymotrypsin, and trypsin, respectively. The fragments contained major parts of the domain controlling the sequence specificity of DNA binding (amino acids 406-514), the SH3 and SH2 domains, and the phosphorylated tyrosine residue Tyr-705, but not the C-terminal 20 amino acids. The N terminus of the 32-kDa tryptic fragment (ANCDASLIV) matched the sequence of amino acids 424-432 deduced from cDNA. The fragments were observed after proteolytic treatment of preformed complexes between DNA and native factors eluted from rat liver nuclei or recombinant, tyrosine-phosphorylated rat Stat3 from insect cells. It was possible to elute all three minimal fragments from their complexes with DNA and to obtain specific re-binding. The minimal fragments eluted from complexes with DNA still contained the phosphorylated Tyr-705 and the SH2 domain suggesting that they were probably bound to DNA as dimers. The DNA-binding domain of Stat3 identified by these experiments overlapped the domain previously identified by genetic experiments as the domain controlling the sequence specificity of DNA binding. The DNA-binding domain defined here by partial proteolysis probably represents an autonomously folding portion of Stat3.

Acute-Phase Proteins↗
PubMed1998

Baculovirus-mediated expression of truncated modular fragments from the catalytic region of human complement serine protease C1s. Evidence for the involvement of both complement control protein modules in the recognition of the C4 protein substrate.

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of complement. Its catalytic region (gamma-B) comprises two complement control protein (CCP) modules, a short activation peptide (ap), and a serine protease domain (SP). A baculovirus-mediated expression system was used to produce recombinant truncated fragments from this region, deleted either from the first CCP module (CCP2-ap-SP) or from both CCP modules (ap-SP). The aglycosylated fragment CCP2-ap-SPag was also expressed by using tunicamycin. The fragments were produced at yields of 0.6-3 mg/liter of culture, isolated, and characterized chemically and then tested functionally by comparison with intact C1s and its proteolytic gamma-B fragment. All recombinant fragments were expressed in a proenzyme form and cleaved by C1r to generate active enzymes expressing esterolytic activity and reactivity toward C1 inhibitor comparable to those of intact C1s. Likewise, the activated fragments gamma-B, CCP2-ap-SP, and ap-SP retained C1s ability to cleave C2 in the fluid phase. In contrast, whereas fragment gamma-B cleaved C4 as efficiently as C1s, the C4-cleaving activity of CCP2-ap-SP was greatly reduced (about 70-fold) and that of ap-SP was abolished. It is concluded that C4 cleavage involves substrate recognition sites located in both CCP modules of C1s, whereas C2 cleavage is affected mainly by the serine protease domain. Evidence is also provided that the carbohydrate moiety linked to the second CCP module of C1s has no significant effect on catalytic activity.

Animals↗
PubMed1999

Selective loss of poly(ADP-ribose) and the 85-kDa fragment of poly(ADP-ribose) polymerase in nucleoli during alkylation-induced apoptosis of HeLa cells.

Alkylation treatment of HeLa cells results in the rapid induction of apoptosis as revealed by DNA laddering and cleavage of poly(ADP-ribose) polymerase (PARP) into the 29-and 85-kDa fragments (Kumari S. R., Mendoza-Alvarez, H. & Alvarez-Gonzalez, R. (1998) Cancer Res. 58, 5075-5078). Here, we performed a time-course analysis of (i) poly(ADP-ribose) synthesis and degradation as well as (ii) the subnuclear localization of PARP and its fragments by using confocal laser scanning immunofluorescence microscopy. PARP was activated within 15 min post-treatment, as revealed by nuclear immunostaining with antibody 10H (recognizing poly(ADP-ribose)). This was followed by a late, time-dependent, progressive decline of 10H signals that coincide with the time of PARP cleavage. Strikingly, nucleolar immunostaining with antibodies 10H and C-II-10 (recognizing the 85-kDa PARP fragment) was lost by 15 min post-treatment, whereas F-I-23 signals (recognizing the 29-kDa fragment) persisted. We hypothesize that the 85-kDa PARP fragment is translocated, along with covalently bound poly(ADP-ribose), from nucleoli to the nucleoplasm, whereas the 29-kDa fragment is retained, because it binds to DNA strand breaks. Our data (i) provide a link between the known time-dependent bifunctional role of PARP in apoptosis and the subcellular localization of PARP fragments and also (ii) add to the evidence for early proteolytic changes in nucleoli during apoptosis.

Alkylation↗
PubMed2000

Insulin inhibits the activation of transcription by a C-terminal fragment of the forkhead transcription factor FKHR. A mechanism for insulin inhibition of insulin-like growth factor-binding protein-1 transcription.

The forkhead rhabdomyosarcoma transcription factor (FKHR) is a promising candidate to be the transcription factor that binds to the insulin response element of the insulin-like growth factor-binding protein-1 (IGFBP-1) promoter and mediates insulin inhibition of IGFBP-1 promoter activity. Cotransfection of mouse FKHR increased IGFBP-1 promoter activity 2-3-fold in H4IIE rat hepatoma cells; insulin inhibited FKHR-stimulated promoter activity approximately 70%. A C-terminal fragment of mouse FKHR (residues 208-652) that contains the transcription activation domain fused to a Gal4 DNA binding domain potently stimulated Gal4 promoter activity. Insulin inhibited FKHR fragment-stimulated promoter activity by approximately 70%. Inhibition was abolished by coincubation with the phosphatidylinositol-3 kinase inhibitor, LY294002. The FKHR 208-652 fragment contains two consensus sites for phosphorylation by protein kinase B (PKB)/Akt, Ser-253 and Ser-316. Neither site is required for insulin inhibition of promoter activity stimulated by the FKHR fragment, and overexpression of Akt does not inhibit FKHR fragment-stimulated Gal4 promoter activity. These results suggest that insulin- and phosphatidylinositol-3 kinase-dependent phosphorylation of another site in the fragment by a kinase different from PKB/Akt inhibits transcription activation by the fragment. Phosphorylation of this site also may be involved in insulin inhibition of transcription activation by full-length FKHR, but only after phosphorylation of Ser-253 by PKB/Akt.

Animals↗
PubMed2001

A protein G fragment from the salmonid viral hemorrhagic septicemia rhabdovirus induces cell-to-cell fusion and membrane phosphatidylserine translocation at low pH.

The fusion-related properties of segments p9, p3, p4, and p9 + p2 surrounding the p2 phospholipid-binding domain of the protein G (pG) of the salmonid rhabdovirus of viral hemorrhagic septicemia (VHS) (Nuñez, E., Fernandez, A. M., Estepa, A., Gonzalez-Ros, J. M., Gavilanes, F., and Coll, J. M. (1998) Virology 243, 322-330; Estepa, A., and Coll, J. M. (1996) Virology 216, 60-70), have been studied at neutral and fusion (low) pH values by using its derived peptides. Cell-to-cell fusion, translocation of phosphatidylserine, and inhibition of fusion of pG-transfected cells defined the p9 + p2 (fragment 11, sequence 56-110) as a fragment with higher specific activity for anionic phospholipid aggregation than the previously reported p2. While fragment 11, p2, and p3 showed interactions with anionic phospholipids, p9 and p4 showed no interactions with any phospholipids. When added to a cell monolayer model at low pH, fragment 11 induced pH-dependent cell-to-cell fusion and translocated phosphatidylserine from the inner to the outer leaflet of the membrane. At low pH and in the presence of anionic phospholipids, fragment 11 showed more than 80% beta-sheet conformation (IR and CD spectroscopies). Finally, anti-fragment 11 antibodies inhibited low pH-dependent pG-transfected cell-to-cell fusion. All of the data support the conclusion that fragment 11 is a primary determinant of some of the viral cell fusion events in VHSV.

Amino Acid Sequence↗
PubMed2003

C3b2-IgG complexes retain dimeric C3 fragments at all levels of inactivation.

C3b2-IgG complexes are formed during complement activation in serum by attachment of two C3b molecules (the proteolytically activated form of C3) to one IgG heavy chain (IgG HC) via ester bonds. Because of the presence of two C3b molecules, these complexes are very efficient activators of the alternative complement pathway. Likewise, dimeric C3b is known to enhance complement receptor 1-dependent phagocytosis, and dimeric C3d (the smallest thioester-containing fragment of C3) linked to a protein antigen facilitates CR2-dependent B-cell proliferation. Because the efficiency of all these interactions depends on the number of C3 fragments, we investigated whether C3b2-IgG complexes retained dimeric structure upon physiological inactivation. We used two-dimensional SDS-PAGE and Western blot to study the arrangement of the C3b molecules by analyzing the fragmentation pattern after cleavage of the ester bonds. Upon inactivation with factors H and I, a 185-kDa band was generated under reducing conditions. It released IgG HC and the 65-kDa fragment of C3b alpha' chain after hydrolysis of the ester bonds with hydroxylamine. The two C3b molecules were not 65-kDa-to-40-kDa linked, because neither ester-bonded 65 kDa HC nor 65 kDa-40 kDa fragments were observed, nor was a 40-kDa peptide released after hydroxylamine cleavage. Factor I and CR1 cleaved the C3b2-IgG molecule to its final physiological product, C3dg2-IgG, which migrated as a 133-kDa fragment in reduced form. This fragment released exclusively C3dg (the final physiological product of C3b inactivation by factor I) and IgG HC. C3dg2-HC appeared as a double band on SDS-PAGE only at low gel porosity, suggesting the presence of two conformers of the same composition. Our results suggest that, upon physiological inactivation, C3b2-IgG complexes retain dimeric inactivated C3b and C3dg, which allows bivalent binding to the corresponding complement receptors.

Blood↗
PubMed1989

Immunological evidences for the presence of small late carboxylterminal fragment(s) of human parathyroid hormone (PTH) in circulation in man.

Two antisera, C-52 and C-97, raised against bovine (b)PTH(1-84) in guinea pigs, were evaluated with 125I-[tyr53] human (h)PTH(53-84) as tracer and intact hPTH(1-84) and synthetic hPTH(39-84), representative of large carboxylterminal ("C") fragments found in circulation, as standards. In both assays, hPTH(39-84) was 5-6 times more potent than hPTH(1-84) on a molar basis in displacing the tracer. With both antisera, progressive deletion at the aminoterminal end of large "C" fragments, as in hPTH(53-84) and hPTH(65-84), lead to decreased immunoreactivity, hPTH(69-84) being non-immunoreactive. The mid-carboxylterminal fragments, hPTH(44-68) and hPTH(39-68), did not react in either assay. Each antiserum measured known quantities of pure hPTH(1-84) or hPTH(39-84) standards similarly. Serum PTH values obtained with antiserum C-97 were about 3 times higher in renal failure, 1.75 times higher in normal individuals and those with primary hyperparathyroidism, while similar to values measured with antiserum C-52 in individuals with secondary hyperparathyroidism without renal failure or with pseudohypoparathyroidism. When circulating PTH taken from patients with these disorders was fractionated by gel chromatography, both antisera recognized similar peaks of intact hPTH(1-84) and of large "C" fragments while antiserum C-97 further recognized a peak of smaller "C" fragments. This explained the different clinical behavior of the latter antiserum. Our findings demonstrate the existence of small late "C" fragments in circulation. They further suggest an influence of serum calcium and of renal function on the quantity of these fragments.

Animals↗
PubMed2000

DNA damage induced by radiation of different linear energy transfer: initial fragmentation.

PURPOSE: To investigate DNA fragmentation as a function of linear energy transfer (LET) after exposure to accelerated ions in the LET range 40-225 keV/microm. MATERIALS AND METHODS: Fragmentation patterns of double-stranded DNA in the range 5 kilobasepairs (kbp) to 5.7 megabasepairs (Mbp) were analysed after irradiation of low-passage GM 5758 normal human fibroblast cells with 60Co-photons, helium ions at 40 keV/microm and high-LET nitrogen ions between 80 and 225 keV/microm. Two separate pulsed-field gel electrophoresis protocols were used, optimized for separation of 1-6 Mbp and 5 kbp to 1.5 Mbp fragments. RESULTS: An increased probability of formation of short and medium-sized DNA fragments was revealed following high-LET irradiation. The DNA double-strand break (dsb) induction yields were, respectively, 5.8 and 6.9-8.8 x 10(-9) dsb bp(-1) Gy(-1) for 60Co-photons and ions. The ion yields were some 80-110% higher than those calculated according to a conventional approach, disregarding the fragment distributions. For photons, the yield was 13% higher. The corresponding relative biological effectiveness (RBE) of dsb induction was in the range 1.2-1.5. CONCLUSIONS: A significant non-random contribution to the number of dsb after irradiation with high-LET was confirmed by detailed fragment analysis using pulsed-field gel electrophoresis. The LET had a strong influence on the initial DNA fragment distribution, and hence also on the induction yields measured. However, when the LET was increased to the highest values studied for nitrogen ions, the yield decreased slightly.

Cells, Cultured↗
PubMed1999

Degraded collagen fragments promote rapid disassembly of smooth muscle focal adhesions that correlates with cleavage of pp125(FAK), paxillin, and talin.

Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125(FAK)), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125(FAK) is a substrate for both calpain I- and II-mediated processing. Mapping of the proteolytic cleavage fragments of pp125(FAK) predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125(FAK) to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125(FAK) with the cytoskeletal fraction, while pp125(FAK) cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125(FAK) cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125(FAK), paxillin, and talin and dissolution of the focal adhesion complex.

Actinin↗
PubMed1983

Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells.

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60-90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.

Animals↗
PubMed2003

Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique.

BACKGROUND: Standard sperm characteristics are poor predictors of the outcome of IVF treatments. On the contrary, sperm genome quality has been emphasized for several years as playing a major role in early embryogenesis, thus in the success of IVF attempt. METHODS: Sperm DNA fragmentation from a selected group of 104 couples undergoing assisted reproductive techniques (ART) (IVF: n = 50; and ICSI: n = 54) was measured by TUNEL assay and correlated with semen and ART outcomes. RESULTS: A negative correlation was found between sperm characteristics and the proportion of sperm showing DNA fragmentation. For fragmentation >10%, a significant decrease of the fertilization rate was observed. No correlation was found between sperm DNA fragmentation and embryo quality. A high proportion of sperm with fragmented DNA was a pejorative factor to obtain pregnancies when ICSI was performed, but there was no relationship when conventional IVF was performed. CONCLUSIONS: The proportion of sperm with DNA fragmentation appears to be potentially useful as a predictor of ICSI outcome, whereas embryo quality based on morphological criteria, appeared unaffected by DNA fragmentation.

DNA Fragmentation↗
PubMed2006

Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART.

BACKGROUND: The relationship between early embryo post-implantation development in couples undergoing assisted reproductive techniques (ARTs) and sperm chromatin alterations has not been satisfactorily explained. The aim of this study was to assess the relationship between sperm DNA fragmentation in IVF/ICSI patients, sperm parameters (concentration, motility and morphology) and ART outcome, especially with regard to clinical pregnancy and pregnancy loss (spontaneous miscarriage or biochemical pregnancy). METHODS: DNA fragmentation was evaluated by TUNEL assay, performed on sperm suspensions after density gradient separation, in 132 men undergoing an ART cycle (82 IVF and 50 ICSI) and correlated with sperm parameters and ART outcome. RESULTS: A highly significant negative correlation was found between DNA fragmentation and sperm parameters. There was a close relationship between DNA fragmentation and post-implantation development in ICSI patients: the clinical pregnancy and pregnancy loss rates significantly differed between patients with high and low sperm DNA fragmentation (P = 0.007 and P = 0.009, respectively). CONCLUSIONS: Sperm DNA fragmentation seems to affect embryo post-implantation development in ICSI procedures: high sperm DNA fragmentation can compromise 'embryo viability', resulting in pregnancy loss.

Abortion, Spontaneous↗
PubMed2003

The soybean beta-conglycinin beta 51-63 fragment suppresses appetite by stimulating cholecystokinin release in rats.

We previously demonstrated that soybean beta-conglycinin peptone suppresses food intake and gastric emptying by direct action on rat small intestinal mucosal cells to stimulate cholecystokinin (CCK) release. The aim of the present study was to define the active fragment in beta-conglycinin by using synthetic peptides chosen from the sequence of three beta-conglycinin subunits. We selected the fragments that had multiple nonadjacent arginine residues, and investigated their ability to bind to components of the rat intestinal brush border membrane as well as to stimulate CCK release and appetite suppression. The fragment from 51 to 63 of the beta subunit (beta 51-63) had the strongest binding activity. Intraduodenal infusion of beta 51-63 inhibited food intake and markedly increased portal CCK concentration. The threshold concentration of beta 51-63 to affect food intake was 3 micro mol/L. The CCK-A receptor antagonist abolished the beta 51-63-induced suppression of food intake. Three types of smaller fragments of beta 51-63 (beta 51-59, beta 53-63 and beta 53-59) and two types of fragments similar to beta 51-63 in the beta-conglycinin alpha and alpha' subunits (alpha 212-224 and alpha' 230-240) had less binding ability than did beta 51-63. Model peptides constructed with arginine (R) and glycine (G), such as GRGRGRG, had strong binding affinity, but peptides containing a single R or RR did not. These results indicate that the beta-conglycinin beta 51-63 fragment is the bioactive appetite suppressant in beta-conglycinin, and multiple arginine residues in the fragment may be involved in this effect.

Animals↗
PubMed1996

Programmed cell death and human embryo fragmentation.

The quality of embryos produced by in-vitro fertilization (IVF) is variable. Many embryos contain unequal sized blastomeres and multiple cellular fragments. Embryos with excessive fragmentation have limited developmental potential both in vitro and in vivo. Histologically, some blastomeres of fragmented embryos resemble cells undergoing apoptosis as a result of programmed cell death (PCD). The objective of the present study was to determine if the morphological features of apoptosis are observed in fragmented human preimplantation embryos, supporting the possible involvement of PCD in early human embryo arrest and demise. Using combined nuclear and terminal transferase-mediated DNA end labelling (TUNEL) on arrested, fragmented human embryos, we were able to detect extensive condensation and degradation of chromatin, compatible with apoptosis. Electron microscopy confirmed the typical morphological features of apoptosis. No such abnormalities were observed in spare embryos with regular sized blastomeres without fragmentation. The high incidence of condensed chromatin, TUNEL detection of degraded DNA, cell corpses and apoptotic bodies in fragmented human embryos strongly suggest that PCD is triggered in human embryos at a stage prior to blastocyst formation. At such early stages, occurrence of apoptosis seemed to be detrimental, leading to preimplantation embryo death.

Apoptosis↗
PubMed1991

Expression of tetanus toxin fragment C in yeast: gene synthesis is required to eliminate fortuitous polyadenylation sites in AT-rich DNA.

Fragment C is a non-toxic 50 kDa fragment of tetanus toxin which is a candidate subunit vaccine against tetanus. The AT-rich Clostridium tetani DNA encoding fragment C could not be expressed in Saccharomyces cerevisiae due to the presence of several fortuitous polyadenylation sites which gave rise to truncated mRNAs. The polyadenylation sites were eliminated by chemically synthesising the DNA with increased GC-content (from 29% to 47%). Synthesis of the entire gene (1400 base pairs) was necessary to generate full-length transcripts and for protein production in yeast. Using a GAL1 promoter vector, fragment C was expressed to 2-3% of soluble cell protein. Fragment C could also be secreted using the alpha-factor leader peptide as a secretion signal. The protein was present at 5-10 mg/l in the culture medium in two forms: a high molecular mass hyper-glycosylated protein (75-200 kDa) and a core-glycosylated protein (65 kDa). Intracellular fragment C was as effective in vaccinating mice against tetanus authentic fragment C. The glycosylated material was inactive, though it was rendered fully active by de-glycosylation.

Adenine↗