Acute up-regulation of brain-derived neurotrophic factor expression resulting from experimentally induced injury in the rat spinal cord.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family of trophic factors, has multiple functions including a role in the promotion of neuronal survival and nerve fiber elongation in both the central and the peripheral nervous systems. We assessed the expression of endogenous BDNF following an experimentally induced compression injury to the spinal cord. Expression of BDNF mRNA was increased following the spinal cord injury; reaching maximum levels 24 h after the injury. Expression of BDNF mRNA returned to the levels observed in sham-operated control animals within 3 days of the injury. Using the in situ hybridization technique, we observed a wide distribution of BDNF expression among the different cell types in the spinal cord, including motor and sensory neurons, and in glia cells, including astrocytes. We also observed expression of BDNF in putative macrophages and/or microglia; however, this effect was not observed until day 7 following spinal cord injury. These results suggest that BDNF is synthesized in both neurons and astrocytes during the acute response to injury to the spinal cord, functioning in a mainly neuroprotective role. This is followed by a later phase of expression in which BDNF is produced by macrophages and/or microglia, apparently functioning in a restorative capacity.

Analysis of effects and pharmacokinetics of subcutaneously administered BDNF.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and has been shown to be a potent and effective trophic factor for motor neurons and other neurons of the peripheral and central nervous. Little is known, however, about the relationship between the efficacy and pharmacokinetics of s.c. administered BDNF. In this study, the efficacy of BDNF on motor neuron protection in sciatic or facial nerve axotomy models was examined and compared with the concommitant concentrations of BDNF in plasma. Delayed treatment (started at 1 week after surgery) of BDNF was also shown to retard choline acetyltransferase reduction in sciatic nerve axotomy models.

BDNF diminishes caspase-2 but not c-Jun immunoreactivity of neurons in retinal ganglion cell layer after transient ischemia.

PURPOSE: Retinal ischemia-reperfusion injury induces apoptosis of retinal neurons. The purpose of this study was to examine the association of c-Jun, caspase-1, -2, and -3 immunoreactivities and neuronal apoptosis in the retinal ganglion cell layer (GCL) and to study the effects of intravitreal brain-derived neurotrophic factor (BDNF) on the expression of these gene products in a rat model of retinal ischemia-reperfusion injury. METHODS: After 60 minutes of ischemia, eyes were enucleated after 3, 6, 12, 24, and 168 hours of reperfusion. The numbers of c-Jun-, caspase-1-, caspase-2-, caspase-3, and TdT-dUTP terminal nick-end labeling (TUNEL)-positive cells in the GCL were counted. Recombinant human BDNF (5 microg) or vehicle was injected intravitreally immediately after reperfusion. At 6, 24, and 168 hours, the numbers of immunoreactive cells in BDNF- and vehicle-treated groups were compared. RESULTS: Expression of c-Jun and caspase-2 was found in dying cells in flat-mounted retinas. The numbers of caspase-1- and caspase-3-positive cells were fewer than c-Jun- or caspase-2-positive cells. Cell death in the retinal GCL was suppressed by an intravitreal injection of BDNF. The numbers of TUNEL- and caspase-2-positive cells were lower in the BDNF-treated group at 6 hours after reperfusion (P<0.01). The number of c-Jun-positive cells in the treated retinas was not altered by the treatment. CONCLUSIONS: Expression of c-Jun and caspase-2 is associated with neuronal cell apoptosis in the GCL. Suppression of caspase-2 expression may explain the neuroprotective effects of BDNF.

Bleomycin enhances random integration of transfected DNA into a human genome.

In mammalian cells, nonhomologous (illegitimate) recombination is a predominant pathway to repair DNA double-strand breaks. We have shown that DNA topoisomerase II inhibitors are capable of enhancing random integration of foreign DNA via nonhomologous recombination. Since this enhancement is likely due to stabilized DNA strand breaks, we examined the effect of a radiomimetic antitumor drug, bleomycin (BLM), on nonhomologous recombination. We found that BLM greatly enhances the random integration of transfected plasmids into human cells. Importantly, this enhancement was independent of the molecular form of the plasmid, the cell type or the transfection method, suggesting that the BLM effect is intrinsically general. Transient expression analysis revealed no stimulation of reporter gene expression by the drug, suggesting that the effect is not attributable to increased uptake and/or accumulation of transfected DNA in the drug-treated cell nuclei. In addition, the comet assay and flow cytometric analyses revealed the occurrence of low but significant strand breaks in cells treated with the BLM concentration which maximally enhanced the integration. These results strongly suggest that BLM acts directly at a nonhomologous recombination reaction that is initiated through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes. Our findings will facilitate the understanding of DNA integration events through nonhomologous recombination and the development of transfection protocols with higher efficiencies.

Construction and characterization of ciliary neurotrophic factor (CNTF) antagonists: microenvironmental difference in the CNTF receptor between rat and chicken cells for recognizing the D1 cap region.

Antagonistic mutants of ciliary neurotrophic factor (CNTF) were constructed and their properties characterized. K155A and K155W mutants lost cell survival promoting activity for chicken dorsal root ganglion (DRG) neurons and inhibited the activity of the wild type. However, they retained slight agonistic activity for the survival of rat DRG neurons, indicating there is a difference between chicken and rat cells for receptor recognition around the D1 cap region including K155 residue. The chicken receptor recognizes the D1 cap region more strictly than does the rat receptor. The substitution of F152, which locates at the top of the D1 cap region, was combined with the K155A mutation. A combination of the two mutations gave an antagonistic feature to not only chicken but also rat cells. Both F152S/K155A and F152D/K155A mutants lacked cell survival promoting activity and had an antagonistic effect on rat DRG neurons. The three-dimensional structure of CNTF suggests the following. F152 and K155 bind to the receptor with hydrophobic and electrostatic interactions, respectively. F152 locates close to L156 with a van der Waals contact, and K155 contacts with Q42 through a hydrogen bond. Both interactions play indispensable roles in maintaining the structure around the D1 cap region of CNTF.

V170 area plays an essential role in the biological activity of human ciliary neurotrophic factor.

The structure-function relationships of human ciliary neurotrophic factor (CNTF) were analyzed by mutagenic means. Amino acid substitutions at helix D caused marked changes in the biological activity of CNTF, suggesting that the residues at helix D of CNTF participate in receptor recognition. In particular, both the cell survival-promoting activity and receptor binding ability of V170 mutant CNTF proteins correlated well with the hydrophobicity of amino acids at position 170. The reduction of hydrophobicity at position 170 resulted in a loss of biological activity, indicating that the hydrophobicity of V170 is essential for the receptor binding and cell survival-promoting activity. Substitutions of R171 or D175 evoked very little folding ability and negated the biological activity of CNTF. As R171 and D175 interact electrostatically with each other and with E75 and R72, respectively, these interactions would be indispensable for stabilizing the whole CNTF protein and for maintaining the structure of the receptor binding epitope.

Activating mechanism of CNTF and related cytokines.

Ciliary neurotrophic factor (CNTF) shares structural and functional properties with members of the hematopoietic cytokine family. It is composed of a four-helix bundle structure and shares the transmembrane signal transducing proteins, glycoprotein-130 (gp130) and leukemia inhibitory factor receptor (LIF-R). Structure-function analysis showed that the gp130-interactive proteins bind in a similar manner to that of growth hormone (site I and II). In addition, gp130-interactive proteins and granulocyte colony-stimulating factor (G-CSF) utilize another binding site (site III) at the boundary between CD loop and helix D. CNTF triggers the association of receptor components, resulting in activation of a signal transduction cascade mediated by specific intracellular protein tyrosine kinases. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and Ras/mitogen-activated protein kinase (MAPK) signaling pathways have been characterized in terms of gp130-interactive protein, and there should be other pathways and some crosstalk between them to enhance, prolong, or specify the signals.

Resident gynecologists and total hysterectomy.

We examined the specific number of surgeries necessary for a three-year obstetrics and gynecology resident to acquire proficiency in two types of hysterectomies. Improvement in the technical skills of the residents was assessed using surgical time and blood loss, and resected tumor weight was chosen as the factor representing the difficulty of the surgery. Regarding abdominal total hysterectomy (ATH), early residents (less than 25 ATH experience) performed relatively easier surgeries, and improvement in technical skill was manifested as reduced blood loss by mid residents (25 to 49 ATH) and as shortened surgical time by later residents (75 or more ATH). Regarding vaginal total hysterectomy (VTH), blood loss for earlier residents (less than 15 VTH) was greater than that for the staff, and there was a significant difference between staff surgical time and that for each resident group. These data suggest that performing more than 75 ATH during the residency period of three years is adequate to establish proficiency in this type of surgery, but that the execution of 25 VTH is insufficient and that residents require more training to learn VTH.

Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor.

We have purified recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in human lymphoblastoid Namalwa cells. From the results of tunicamycin treatment and N-glycosidase F digestion, it was demonstrated that Namalwa-derived hGM-CSF was highly glycosylated at two potential N-glycosylation sites and several O-glycosylation sites as previously shown for naturally occurring hGM-CSF. We classified the hGM-CSF molecules into three groups according to the molecular weight corresponding to the degree of N-glycosylation: the molecules with two N-glycosylation sites occupied (designated 2N), the molecules with either site glycosylated (1N), and the molecules lacking N-glycosylation (0N). Despite such varied degrees of N-glycosylation, almost all molecules were O-glycosylated. To investigate the role of carbohydrate moieties of hGM-CSF, we isolated each form of hGM-CSF and examined its biological properties. The 2N-type showed 200-fold less in vitro specific activity compared with unglycosylated Escherichia coli-derived hGM-CSF, although the activity of the 0N-type was equivalent to that of the E. coli-derived material. The 1N-type showed an intermediate level of activity. However, in terms of clearance from blood circulation in the rat, the 2N-type showed a half-life five times longer than that of the 0N-type and E. coli-derived hGM-CSF. From these findings, we concluded that N-linked carbohydrate moieties of hGM-CSF play conflicting physiological roles in the efficacy of the protein in vivo but that O-linked carbohydrate moieties do not have such effects.

Amplification and high-level expression of a cDNA for human granulocyte-macrophage colony-stimulating factor in human lymphoblastoid Namalwa cells.

We have achieved high-level expression of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in human lymphoblastoid Namalwa cells by introducing and subsequently amplifying an expression vector encoding human GM-CSF and mouse dihydrofolate reductase (DHFR). Transformants expressing elevated levels of both GM-CSF and DHFR were selected by a step-wise increase in the concentration of methotrexate (MTX). Several cell lines resistant in 800 nM MTX have been established that express GM-CSF at a rate of 10-20 micrograms/10(6) cells/day. The amplified genes are integrated and stably maintained in the chromosomes of these cell lines. Analysis of the GM-CSF produced in the amplified Namalwa cells revealed that the molecular-size distribution due to varied degrees of glycosylation was similar to that observed in the naturally-occurring molecules.

Structure-function relationships in the transposition protein B of bacteriophage Mu.

The B-protein of phage Mu, which is required for high frequency intermolecular transposition in vivo, shows ATPase activity in vitro, binds nonspecifically to DNA, and stimulates intermolecular strand transfer. To elucidate the structural bases for B-protein function, it was subjected to limited proteolysis with two different proteases, trypsin and chymotrypsin. The resulting fragments were mapped by amino acid sequencing. These data show that the B-protein is organized in two domains: an amino-terminal domain of 25 kDa and a carboxyl-terminal domain of 8-kDa. A fragment analogous to the amino-terminal domain, produced by deleting the 3' end of a cloned B gene, proved to be insoluble and had to be renatured after elution from a sodium dodecyl sulfate gel. The renatured protein retains ATP-binding activity and to a lesser extent the DNA-binding activity of the MuB protein, but is unable to hydrolyze ATP or function in transposition. We also show in this study that efficient DNA-strand transfer by the B-protein occurs even in the absence of a detectable ATPase activity or in the presence of adenosine 5'-O-(thio)triphosphate (ATP gamma S).

Unusual bone scintigraphy in chronic myelogenous leukemia--report of a case showing extensive uptake defect.

An extensive 99mTc-methylene diphosphonate uptake defect was observed on bone scintigraphy in a 35-year-old male with chronic myelogenous leukemia. This type of bone scintigraphy pattern is quite unusual in leukemic patients and we speculate that acute disturbance of blood supply to the bone marrow was probably the cause.

[Bone scintigraphy in bone metastases due to prostatic cancer].

Findings of bone scintigraphy with 99mTc-MDP were compared with bone radiography and biochemical data including total acid phosphatase (T. ACP), prostatic acid phosphatase (P. ACP), and alkaline phosphatase (ALP) in 35 patients with histologically proven prostatic cancer. Bone metastases were diagnosed in 20 of 35 cases (57%) by scintigraphy. The common sites of metastases were the pelvic bones, ribs, lumbar and thoracic vertebrae. In vertebrae, metastases were mainly distributed in the lower level. The most frequent radiographic change due to metastases was the osteoblastic type. On follow-up studies, there was a relatively good agreement in the results of bone scintigraphy and radiography. However, there was a good number of cases showing discrepancy between either scintigraphy or radiography and laboratory data. Bone scintigraphy seems to be the most contributory in monitoring bone metastases from prostatic cancer.