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PubMed2006

Effect of post-hypoxic reoxygenation on DNA fragmentation in cortical neuronal nuclei of newborn piglets.

Previous studies have shown an increased fragmentation of genomic DNA following hypoxia in cortical neuronal nuclei of newborn piglets. The present study tests the hypothesis that DNA fragmentation following hypoxia persists during reoxygenation in cortical neuronal nuclei of newborn piglets. To test this hypothesis, DNA fragmentation was assessed in 36 newborn piglets divided into six groups: normoxic (Nx), hypoxic (Hx) and hypoxic/reoxygenated for 6, 12, 24h and 7 days. The Hx groups were exposed to 7% oxygen for 1h followed by reoxygenation to room air for 6, 12, 24h and 7 days. Cerebral tissue hypoxia was confirmed biochemically by ATP and phosphocreatine (PCr) levels. Nuclei were isolated and purified using discontinuous sucrose gradient. DNA was isolated by phenol/chloroform/isoamyl-alcohol extraction method. ATP/PCr (micromol/g brain) were 4.11+/-0.15/3.67+/-0.30 for Nx, 1.31+/-0.68/0.74+/-0.30 for Hx, 3.81+/-0.11/3.24+/-0.14 for 6h reoxygenation, 4.21+/-0.12/3.27+/-0.09 for 12h reoxygenation and 4.63+/-0.09/3.75+/-0.27 for 24h reoxygenation and 4.31+/-0.12/3.70+/-0.21 for 7 days reoxygenation. There was a significant difference in the ATP and PCr values between Nx and Hx groups (p<0.05) and between Hx and hypoxic reoxygenated groups (p<0.05). DNA fragments (OD/mm(2)) increased from 1776+/-267 in the Nx group to 3211+/-285 in the Hx group (p<0.05). In the reoxygenation groups, DNA fragments (OD/mm(2)) decreased to 2018+/-249 after 6h (p<0.05 versus Hx) but increased to 3408+/-206, 2782+/-406 and 3256+/-302 after 12, 24h and 7 days, respectively. The data show a decrease in DNA fragmentation in the early phase (6h) of reoxygenation but is comparable to acute hypoxia during the later phases (12, 24h and 7 days) of reoxygenation. We propose that the biphasic pattern of DNA fragmentation during reoxygenation occurs by an initial oxidative DNA injury followed by an enzymatic cleavage of DNA by endonucleases activation.

Adenosine Triphosphate↗
PubMed2006

Hypoxia-induced Bax and Bcl-2 protein expression, caspase-9 activation, DNA fragmentation, and lipid peroxidation in mitochondria of the cerebral cortex of newborn piglets: the role of nitric oxide.

The present study tests the hypothesis that cerebral hypoxia results in increased ratio of Bax/Bcl-2, activation of caspase-9, lipid peroxidation, and DNA fragmentation in mitochondria of the cerebral cortex of newborn piglets and that the inhibition of nitric oxide synthase by N-nitro-L-arginine during hypoxia will prevent the events leading to mitochondrial DNA fragmentation. To test this hypothesis, six piglets, 3-5 days old, were divided into three groups: normoxic (n=5), hypoxic (n=5), and hypoxic-nitric oxide synthase (n=4). Hypoxic animals were exposed to a FiO2 of 0.6 for 60 min. Nitric oxide synthase (40 mg/kg) was infused over 60 min prior to hypoxia. Tissue hypoxia was confirmed by measuring levels of ATP and phosphocreatine. Cerebral cortical tissue mitochondria were isolated and purified using a discontinuous ficoll gradient. Mitochondrial Bax and Bcl-2 proteins were determined by Western blot. Caspase-9 activity in mitochondria was determined spectro-fluorometrically using fluorogenic substrate for caspase-9. Fluorescent compounds, an index of mitochondrial membrane lipid peroxidation, were determined spectrofluorometrically. Mitochondrial DNA was isolated and separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. ATP levels (micromol/g brain) were 4.52+/-0.34 in normoxic, 1.18+/-0.29 in hypoxic (P<0.05) and 1.00+/-0.26 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic). Phosphocreatine levels (micromol/g brain) were 3.61+/-0.33 in normoxic, 0.70+/-0.20 in hypoxic (P<0.05 vs. normoxic) and 0.57+/-0.14 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic, P=NS vs. hypoxic). Bax density in mitochondrial membranes was 160+/-28 in normoxic and 324+/-65 in hypoxic (P<0.001 vs. normoxic). Bcl-2 density mitochondria was 96+/-18 in normoxic and 98+/-20 in hypoxic (P=NS vs. normoxic). Mitochondrial caspase-9 activity (nmol/mg protein/h) was 1.32+/-0.23 in normoxic and 2.25+/-0.24 in hypoxic (P<0.01 vs. normoxic). Levels of fluorescent compounds (microg of quinine sulfate/g protein) were 12.48+/-4.13 in normoxic and 37.92+/-7.62 in hypoxic (P=0.003 vs. normoxic). Densities (ODxmm2) of low molecular weight DNA fragments were 143+/-38 in normoxic, 365+/-152 in hypoxic, (P<0.05 vs. normoxic) and 163+/-25 in hypoxic-nitric oxide synthase animals (P<0.05 vs. hypoxic, P=NS vs. normoxic). The data demonstrate that hypoxia results in increased mitochondrial proapoptotic protein Bax, increased mitochondrial caspase-9 activity, increased mitochondrial lipid peroxidation, and increased fragmentation of DNA in mitochondria of the cerebral cortex of newborn piglets. The administration of a nitric oxide synthase inhibitor, nitric oxide synthase, prior to hypoxia prevented fragmentation of mitochondrial DNA, indicating that the hypoxia-induced mitochondrial DNA fragmentation is NO-mediated. We propose that NO free radicals generated during hypoxia lead to NO-mediated altered expression of Bax leading to increased ratio of pro-apoptotic/anti-apoptotic protein resulting in modification of mitochondrial membrane, and subsequently Ca2+-influx and fragmentation of mitochondrial DNA.

Adenosine Triphosphate↗
PubMed2005

Circulating fragmented nucleosomal DNA and caspase-3 mRNA in patients with lymphoma and myeloma.

Elevated amounts of cell-free nucleic acids are detected in the circulation of cancer patients. The type and pattern of these may vary depending on the origin. Recently, we described the presence of circulating fragmented nucleosomal DNA. In the present study, our aim was to investigate the association between nucleosomal DNA, caspase-3 expression and circulating caspase-3 mRNA, the primary activator of nucleosomal DNA fragmentation. DNA fragmentation was analyzed by gel electrophoresis, and caspase-3 was analyzed by RT-PCR. Plasma samples from 65% of patients were positive for nucleosomal DNA fragmentation, whereas none of the healthy controls showed DNA fragmentation (P = 0.0004). Expression of caspase-3 differed significantly between the cases and controls (P < 0.0001). However, we observed no direct correlation between nucleosomal DNA fragmentation and caspase-3 expression in lymphocytes (P = 0.145). Circulating plasma caspase-3 mRNA was detected by nested RT-PCR, and no significant difference was observed between the patients and the control group (P = 0.5). Our results indicate that caspase-3 expression is increased in lymphocytes from patients. When compared to healthy individuals, no differences were observed in the amount of circulating mRNA. These findings suggest that nucleosomal DNA fragmentation is not correlated with elevated levels of apoptosis and circulating caspase-3 mRNA in circulating tumor cells.

Adult↗
PubMed1998

Midtrimester urine human chorionic gonadotropin beta-subunit core fragment levels and the subsequent development of pre-eclampsia.

OBJECTIVE: Our purpose was to determine whether midtrimester maternal urine human chorionic gonadotropin beta-subunit core fragment predicts later pre-eclampsia. STUDY DESIGN: Urine beta-core fragment levels standardized to spot creatinine concentration and expressed as multiples of the median were prospectively determined in 347 midtrimester singleton pregnancies undergoing genetic amniocentesis. All women considered in the analysis were white and nonsmokers. Obstetric chart review was undertaken after delivery to identify cases in which pre-eclampsia developed. The risk of pre-eclampsia at different threshold levels of beta-core fragment of human chorionic gonadotropin was determined. RESULTS: The median maternal age was 36.0 years, with a median gestational age at urine collection of 16.0 weeks. The median level of the beta-core fragment of human chorionic gonadotropin was 1385.5 ng/mg of creatinine in those with pre-eclampsia, whereas that in those without pre-eclampsia was 1061.2 ng/mg. The difference was significant (Mann-Whitney U test, P = .03). A significant linear association was found between the beta-core fragment concentration and the risk of pre-eclampsia (Mantel-Haenszel test of linear association, P = .03). The relative risk and 95% confidence interval of subsequent pre-eclampsia increased from 2.07 (1.06 to 4.05) at beta-core fragment levels of human chorionic gonadotropin > or = 2.0 multiples of the median to 5.17 (1.95 to 13.7) at > or = 4.0 multiples of the median. CONCLUSION: Clinically normal patients with elevated midtrimester levels of urine beta-core fragment of human chorionic gonadotropin are at increased risk for the subsequent development of pre-eclampsia. The clinical value of this urine analyte as a marker for pre-eclampsia needs to be further investigated.

Adult↗
PubMed1997

Inhibition of cross-bridge binding to actin by caldesmon fragments in skinned skeletal muscle fibers.

Several regions within the 35-kDa COOH-terminal portion of caldesmon have been implicated in the ability of caldesmon to inhibit actin-activated myosin ATPase activity. To further define the functional regions of caldesmon, we have studied the effects of three chymotryptic fragments, one fragment produced by CNBr digestion and two fragments produced by digestion with submaxillaris arginase C protease, on the relaxed stiffness and active force of rabbit psoas fibers. Each of the regions of caldesmon studied had either direct or indirect effects on single-fiber mechanics. The 35-kDa and 20-kDa fragments of caldesmon, like intact caldesmon, were effective inhibitors of fiber stiffness, a measure of cross-bridge attachment. The 7.3-kDa and 10-kDa fragments, which constitute the NH2 and COOH halves of the 20-kDa fragment, inhibited both relaxed fiber stiffness and active force production, but with a reduced efficacy compared to the 20-kDa fragment. These results suggest that several regions within the 35-kDa COOH-terminal region of caldesmon are required for optimum function of caldesmon and that function includes inhibition of weak cross-bridge attachment and force production.

Actins↗
PubMed2002

Internucleosomal DNA fragmentation during deprived and non-deprived olfactory development.

DNA fragmentation is a key marker of neuronal death during development, yet little is known about the size, pattern or quantities of fragments generated during normal and sensory-deprived development. Since there are few neurons dying at any particular time, it has not been possible to obtain sufficient quantities of material to make such a determination. By using a highly sensitive Taq polymerase-based technique, we revealed DNA fragments of 180 base pairs and multiples thereof both in bulbs and cortex of young rats (P4-P31). The bulbs subjected to olfactory deprivation at P1 had higher levels of internucleosomal DNA fragmentation at P16 than the contra-lateral, non-deprived bulbs. Interestingly, the DNA fragmentation induced by olfactory deprivation displayed a characteristic internucleosomal fragmentation pattern, suggesting that the cells induced to die may do so by apoptosis. A significant inverse correlation between DNA fragmentation and the natural variation in normal bulb size was found, suggesting that bulb size may be related to cell death.

Animals↗
PubMed2000

Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin.

OBJECTIVES: This study investigates the sensitivity of various standard clinical techniques in the detection of albumin fragments. The significance of this work is in the detection of urinary proteins, such as albumin, which has recently been discovered to be excreted as mainly peptide fragments as a result of filtered albumin undergoing degradation during renal passage. All filtered proteins undergo a similar degradation process. DESIGN AND METHODS: Albumin digested with trypsin was used as a model urine solution. The solution was assayed for albumin concentration by various methods including the biuret assay that is known to detect urinary albumin fragments. The digest solution was also analyzed by various clinically used chromagen assays, electrophoretic and chromatographic methods to determine whether they are able to detect the fragmented protein. RESULTS: The benzethonium chloride, Coomassie blue, and pyrogallol red assays for urine protein, the immunoassay for human albumin and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining were unable to detect the albumin fragments. Capillary electrophoresis was sensitive to the fragments but with low resolution. High-performance liquid chromatography gave the best results. CONCLUSIONS: Many techniques utilized to assay patient urine samples are unable to detect fragmented albumin and, hence, will severely underestimate albumin and protein excretion.

Albuminuria↗
PubMed1999

Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria.

The equilibrium denaturation and unfolding kinetics of the domains V(L) and V(H) have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimeric assembly even further. Domain dissociation and domain unfolding are coupled processes, giving rise to a highly cooperative unfolding transition. For the Fv fragment, cooperative unfolding is only observed in the presence of antigen. At low protein concentrations and in the absence of antigen, the Fv fragment is significantly destabilized, leading to quantitative domain dissociation before significant domain unfolding takes place. The kinetic unfolding of V(H), V(L) and the scFv fragment is monophasic. Unfolding of the scFv fragment is much slower, when extrapolated to zero denaturant, than either of the isolated domains, suggesting that the higher thermodynamic stability of the scFv fragment is at least partially due to a high-energy transition state for unfolding. These studies emphasize the enormous importance of mutual domain stabilization in engineering stable antibodies.

Dose-Response Relationship, Drug↗
PubMed2002

Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep.

OBJECTIVE: To evaluate DNA fragmentation in the oocyte of primordial and primary follicles and morphology of these follicles after freezing and thawing of ovarian cortex in sheep using two freezing protocols. DESIGN: Fragmentation of DNA was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique. SETTING: Fertility clinic in a large university hospital. ANIMALS: Five- to 6-month-old lambs. INTERVENTION(S): Two-millimeter-thick slices of hemi-ovary cortex were prepared. MAIN OUTCOME MEASURE(S): Histological structure and DNA fragmentation. RESULT(S): In the frozen fragments, the percentage of morphologically normal follicles was significantly lower for both protocols compared with the case of the control group of fresh fragments. There was no significant difference between the two types of freezing protocols (60.4% +/- 13.2% vs. 68.4% +/- 13.7%). However, the distribution of abnormalities (nucleus, cytoplasm, and nucleus and cytoplasm) was dissimilar. The results of the TUNEL technique for the three groups showed no significant difference, but the percentage of the TUNEL-positive follicles was slightly lower for the frozen fragments for both protocols with respect to the control group. CONCLUSION(S): The freezing and thawing process of the ovarian cortex does not induce fragmentation of the DNA on the oocyte of primary and primordial follicles.

Animals↗
PubMed1998

Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection.

OBJECTIVE: To determine the incidence of DNA fragmentation in human sperm used for intracytoplasmic sperm injection (ICSI) and to correlate any detected DNA damage with semen analysis parameters and fertilization rates in ICSI. DESIGN: Descriptive and correlational clinical study. SETTING: Tertiary care fertility clinic. PATIENT(S): A total of 150 semen samples was collected from men in the ICSI program. INTERVENTION(S): For each sample, sperm wash and swim-up were performed, and the percentage of recovered sperm with DNA fragmentation was determined with the use of terminal transferase-mediated deoxyuridine triphosphate-biotin end labeling. MAIN OUTCOME MEASURE(S): The percentage of sperm with DNA fragmentation was correlated with semen analysis parameters and ICSI fertilization rates. RESULTS(S): The mean (+/- SD) percentage of sperm with fragmented DNA was 14.5% +/- 1.5% and ranged from 0.5% to 75%. A significant negative association was found between the percentage of sperm with DNA fragmentation and the ICSI fertilization rate. We also observed that the motility and morphology of the ejaculated sperm were correlated negatively with the percentage of DNA fragmentation in the washed sperm recovered by the swim-up technique. CONCLUSION(S): Our results suggest that when poor-quality semen samples are used for ICSI, there is a greater likelihood that some sperm selected for injection, despite appearing normal, contain fragmented DNA. Whether sperm DNA damage may contribute to failure of pronuclear formation and embryo development in some apparently unfertilized ICSI oocytes is unclear.

Cleavage Stage, Ovum↗
PubMed2003

Fourier transform infrared spectroscopy used to evidence the prevention of beta-sheet formation of amyloid beta(1-40) peptide by a short amyloid fragment.

Reflectance Fourier transform infrared (FT-IR) microspectroscopy was applied to study the prevention of beta-sheet formation of amyloid beta (Abeta)(1-40) peptide by co-incubation with a hexapeptide containing a KLVFF sequence (Abeta(15-20) fragment). Second-derivative spectral analysis was used to locate the position of the overlapping components of the amide I band of Abeta peptide and assigned them to different secondary components. The result indicates that each intact sample of Abeta(15-20) fragment or Abeta(1-40) peptide previously incubated in distilled water at 37 degrees C transformed their secondary structure from 1649 (1651) or 1653cm(-1) to 1624cm(-1), suggesting the transformation from alpha-helix and/or random coil structures to beta-sheet structure. By co-incubating both samples with different molar ratio in distilled water at 37 degrees C, the structural transformation was not found for Abeta(1-40) peptide after 24h-incubation. But the beta-sheet formation of Abeta(1-40) peptide after 48h-incubation was evidenced from the appearance of the IR peak at 1626cm(-1) by adding a little amount of Abeta(15-20) fragment. There was no beta-sheet formation of Abeta(1-40) peptide after addition with much amount of Abeta(15-20) fragment, however, suggesting the higher amount of Abeta(15-20) fragment used might inhibit the beta-sheet formation of Abeta(1-40) peptide. The more Abeta(15-20) fragment used made the more stable structure of Abeta(1-40) peptide and the less beta-sheet formation of Abeta(1-40) peptide. The study indicates that the reflectance FT-IR microspectroscopy can easily evidence the prevention of beta-sheet formation of Abeta(1-40) peptide by a short amyloid fragment.

Amyloid beta-Peptides↗
PubMed1997

Phage-displayed and soluble mouse scFv fragments neutralize rabies virus.

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (VH) and light (VL) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.

Amino Acid Sequence↗
PubMed1999

Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies.

Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and their antigen is close to the nanomolar range, similar to the bivalent mouse monoclonal antibodies studied. Llama VHH antibody fragments are similar to mouse monoclonal antibodies with respect to antigen binding in the presence of ammonium thiocyanate and ethanol. The results show that relative to antigen specific mouse monoclonal antibodies, antigen specific llama VHH fragments are extremely temperature stable. Two out of six llama VHHs are able to bind antigen specifically at temperatures as high as 90 degrees C, whereas four out of four mouse monoclonal antibodies are not functional at this temperature. Together with the finding that llama VHH fragments can be produced at high yield in Saccharomyces cerevisiae, these findings indicate that in the near future antigen specific llama VHH fragments can be used in for antibodies unexpected products and processes.

Animals↗
PubMed1999

Autonomous folding of a C-terminal inhibitory fragment of Escherichia coli isoleucine-tRNA synthetase.

We previously reported that C-terminal fragments of Escherichia coli Ile-tRNA synthetase, a monomeric enzyme of 939 amino acids, act as dominant negative inhibitors of the wild-type enzyme in vivo and in vitro. Our experiments suggested that it is possible to block the functional assembly of a monomeric protein by interfering with the folding pathway. We postulated that the inhibitory C-terminal fragments fold autonomously, and in the presence of full-length Ile-tRNA synthetase, trap the N-terminal portion of polypeptide in an unproductive complex. Here, we report the results of experiments aimed at understanding the mechanism of dominant negative inhibition. We have carried out biophysical experiments on fragment 585-939 of Ile-tRNA synthetase, which we previously determined to be the minimal inhibitory unit. Circular dichroism and fluorescence spectroscopy indicate that this fragment forms a compact and stable structure in solution. The secondary structure of this fragment is predominantly alpha-helical, consistent with the crystal structure of Ile-tRNA synthetase from another organism. The C-terminal fragment is capable of forming native-like secondary and tertiary structure after refolding from guanidine HCl. Taken together, the results are consistent with the hypothesis that the inhibitory fragment of Ile-tRNA synthetase forms an independent folding unit.

Circular Dichroism↗
PubMed2001

DNA fragmentation in ischemic core and penumbra in focal cerebral ischemia in rats.

Although apoptotic cell death has been suggested to be involved in ischemic injury of the brain, the precise mechanisms of ischemic neuronal cell death are unknown. Here, we examined the biochemical feature of apoptosis (i.e. DNA fragmentation) in male spontaneously hypertensive rats (5-7 months old) subjected to photothrombotic distal middle cerebral artery (MCA) occlusion. After MCA occlusion, the brain was cut in a cryostat to produce a standard coronal block and samples were dissected from the regions corresponding to the ischemic core, penumbra and contralateral control areas. Changes in cerebral blood flow (CBF) were monitored at 1 mm posterior and 2-4 mm lateral to the bregma by means of a laser-Doppler flowmetry. After MCA occlusion, CBF was decreased to 72+/-18 (+/-S.D.), 50+/-14, and 35+/-11% of the control values at 2, 3, and 4 mm from the midline, respectively. DNA fragmentation characteristics of apoptosis were examined in these samples by conventional and pulse-field gel electrophoresis. On the conventional gel electrophoresis, nucleosomal DNA fragmentation was detected in the penumbral zone at 6 h after MCA occlusion. Large DNA fragments of 50 and 20 kbp were detected in the penumbral zone and also in the ischemic core region at 3 h after distal MCA occlusion. The large DNA fragments seen on the pulse-field gel elecrophoresis were further degraded to small DNA fragments at 6 h after MCA occlusion in the penumbral zone but not in the core regions. The evolving DNA fragmentation was observed between 3 and 6 h after the onset of brain ischemia in the penumbra, suggesting that apoptosis may contribute to the development of ischemic infarction.

Animals↗
PubMed1997

The effect of naturally occurring fragments of galanin message-associated peptide on spinal cord excitability in rats.

Galanin message-associated peptide (GMAP), a 60 amino acid fragment of galanin precursor protein, is present in dorsal root ganglion cells and upon intrathecal (i.t.) administration influences the spinal nociceptive flexor reflex in rat in a complex manner. The present study assessed the effects on spinal cord excitability of N-terminal fragment GMAP (1-30) and C-terminal fragments GMAP (34-60) or GMAP (35-60), which were formed from GMAP following enzymatic degradation. The effect of the fragments was compared with the effects of the complete peptide sequence. The GMAP fragments slightly facilitated the flexor reflex and dose-dependently blocked hyperexcitability following C-fiber conditioning stimulation. The potency of the blocking effect of GMAP (1-30) was comparable to GMAP (1-60) and was one order of magnitude higher than the potency of the C-terminal fragments. The results indicated that both naturally formed N- and C-terminal fragments of GMAP are pharmacologically active and produce effects which are similar to the full peptide sequence.

Animals↗
PubMed1999

Effects of an acidic fibroblast growth factor fragment analog on learning and memory and on medial septum cholinergic neurons in senescence-accelerated mice.

We examined the effects of repeated subcutaneous injections of an acidic fibroblast growth factor fragment analog, [Ala16] acidic fibroblast growth factor (1-29), on learning and memory and on the choline acetyltransferase immunoreactivity of forebrain neurons in senescence-accelerated mice. One group of accelerated senescence-prone mice (accelerated senescence-prone-8) received [Ala16] acidic fibroblast growth factor (1-29), whereas the other group of accelerated senescence-prone-8 mice and a group of accelerated senescence-resistant mice (control) received vehicle solution. Injections began at three weeks after birth and were given weekly for 10 months. In a passive avoidance test, the mean retention latency at three, six and nine months of age was significantly longer in controls (vehicle-treated accelerated senescence-resistant-1) and acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 than in vehicle-treated accelerated senescence-prone-8 mice, and the latency in acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice was significantly shorter than that in controls only at nine months of age. In the Morris water maze task, the mean latency to climb onto the platform was significantly longer in acidic fibroblast growth factor fragment- and vehicle-treated accelerated senescence-prone-8 mice than in controls. However, the mean latency in the third and fourth trial blocks was significantly shorter for acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 than for vehicle-treated accelerated senescence-prone-8 mice. In the probe trials, controls and acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice spent significantly more time in the quadrant in which the platform had previously been located than in the other three quadrants. In acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice, the density of medial septum neurons intensely stained for choline acetyltransferase was significantly greater than that in vehicle-treated accelerated senescence-prone-8 mice, but significantly less than that in controls. The results indicate that the beneficial effect of [Ala16] acidic fibroblast growth factor (1-29) on learning and memory function in accelerated senescence-prone-8 mice may be related to a preservation of function in medial septum cholinergic neurons.

Aging↗
PubMed1999

The time-course of DNA fragmentation in the choroid plexus and the CA1 region following transient global ischemia in the rat brain. The effect of intra-ischemic hypothermia.

The time-course of DNA fragmentation in the CA1 region of the hippocampus and the choroid plexus was studied following induction of transient forebrain ischemia under lethal normothermic (37 degrees C), or sublethal hypothermic (33 degrees C) conditions. Oligonucleosomal- and high-molecular-weight DNA fragmentation were analysed by conventional agarose gel electrophoresis and pulsed-field gel electrophoresis, respectively. DNA breaks were visualized by the terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridinetriphosphate nick-end labeling method. At 48 h of recovery following normothermic ischemia, in situ labeling of DNA breaks were widespread in medial CA1 and high-molecular-weight DNA cleavage was seen. In contrast, at the same time-point in lateral CA1, many pyknotic but few cells displaying in situ labeling of DNA breaks were observed. Major oligonucleosomal DNA fragmentation was not seen until 72 h of recovery. Following hypothermic ischemia, DNA fragmentation was absent in CA1. DNA fragmentation was seen in the choroid plexus at 24 h of recovery following normothermic ischemia, which was diminished by 48 h of recovery. In conclusion, oligonucleosomal and high-molecular-weight DNA fragmentation at 10-50 kilobase pairs, occur in CA1 after morphological signs, and acidophilia signifying neurodegeneration appear. DNA fragmentation and cell death in the choroid plexus precede neuronal death in CA1 and may play a causative role.

Animals↗