Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats.

Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) causes cerebral ischemia and infarction. To date, the pathogenesis and gene expression associated with vasospasm remain poorly understood. The present study used fluorescent differential display to identify differentially expressed genes in a rat model of SAH. By using quantitative RT-PCR, we found that heme oxygenase-1 (HO-1) mRNA was prominently induced in the basilar artery and modestly in brain tissue in a rat vasospasm model. A significant correlation was observed between the degree of vasospasm and HO-1 mRNA levels in the basilar arteries exhibiting vasospasm. Intracisternal injection of antisense HO-1 oligodeoxynucleotide (ODN) significantly delayed the clearance of oxyhemoglobin and deoxyhemoglobin from the subarachnoid space and aggravated angiographic vasospasm. Antisense HO-1 ODN inhibited HO-1 induction in the basilar arteries but not in the whole brain tissue. This phenomenon was not observed in the nontreated, sense HO-1 ODN-treated, or scrambled ODN-treated arteries. We report the protective effects of HO-1 gene induction in cerebral vasospasm after SAH, a finding that should provide a novel therapeutic approach for cerebral vasospasm.

Cyclin-dependent kinase inhibitor p21 modulates the DNA primer-template recognition complex.

The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, we have designed a new assay for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPgammaS-activated complex was no longer displaced from the DNA by p21. We propose that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome.

Nonhistone proteins HMG1 and HMG2 suppress the nucleosome assembly at physiological ionic strength.

The effect of nonhistone protein HMG1 and HMG2 from pig thymus on the in vitro nucleosome assembly has been examined with plasmid pSV2-gpt DNA and pig thymus core histones in the presence of DNA topoisomerase I. In the absence of core histones, the direct binding of HMG proteins could induce negative superhelical turns in DNA at low ionic strength, but not at physiological ionic strength. The nucleosome formation in the higher histone-to-DNA ratios at physiological ionic strength was not facilitated by HMG proteins, in contrast to poly(L-glutamic acid). HMG proteins suppressed the nucleosome assembly in the moderate histone-to-DNA ratios, resulting in the reduction of fully supercoiled DNA topoisomers. The suppression by HMG proteins was not cancelled by poly(L-glutamic acid). These suggest that the highly acidic carboxy terminal of HMG proteins does not act as an assembly factor, and that the HMG proteins, on the contrary, suppress the nucleosome formation, probably by binding to DNA in a way to inhibit the assembly into core particles.

Determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by Bombyx mori.

The analysis of fibroin secretion-deficient 'naked-pupa' mutant silkworms has suggested that the disulfide linkage between heavy (H) and light (L) chains of fibroin, produced by the silkworm, Bombyx mori, is essential in its efficient large-scale secretion from the posterior silk gland cells. However, the site of disulfide-linkage between H- and L-chains has not been determined. In this study, cysteine residues involved in the single disulfide linkage between H- and L-chains were identified as the twentieth residue from the carboxyl terminus of H-chain (Cys-c20) and Cys-172 of L-chain by sequencing of genomic clones and peptide analysis. Furthermore, Cys-c4 (fourth residue from the carboxyl terminus) and Cys-c1 at the carboxyl terminus of H-chain were shown to form an intramolecular disulfide bond.

Production of a chimeric fibroin light-chain polypeptide in a fibroin secretion-deficient naked pupa mutant of the silkworm Bombyx mori.

The allelic Nd-s and Nd-sD mutations of the silkworm Bombyx mori are mapped to the same locus as that of the fibroin light (L)-chain gene (Fib-L). The silkworm carrying the homozygous Nd-s or Nd-sD mutation secretes less than 0.3% of the normal level of fibroin and produces a thin cocoon (naked pupa). In this study, cDNA sequences of the Nd-s and Nd-sD L-chains were compared with the cDNA and genomic sequences of the L-chain of the B. mori J-139 strain, a normal-level producer of fibroin. The two mutant cDNA sequences are almost identical except for one base change in the coding region. The N-terminal half of the L-chain encoded by exons I to III is identical between the mutants and J-139, but the rest of the molecule is completely different. The C-terminal half of the Nd-sD mutant L-chain is encoded by two exons, IV' and V', which are brought into proximity with the exon III by recombination between sequences in the third intron and in the far downstream region with concomitant loss of a region containing exons IV to VII. Sequences corresponding to exons IV' and V' are present about 10 kb downstream of the L-chain gene in the J-139 genome. Their homologous sequences have not been found in the DNA and protein databases. The chimeric L-chain molecule of about 27 kDa is present in posterior silk glands of Nd-s and Nd-sD strains without disulfide-bonding to the fibroin heavy (H-) chain, as revealed by Western blotting with the antibody specific to the C-terminal half of the mutant L-chain.

p53-induced p21 controls DNA replication.

The p21 protein, a regulator of cyclin-dependent kinases (CDKs), has been thought to be one of the key proteins to function in cell proliferation suppression upon DNA damage. In normal cells but not in many tumor cells, p21 forms a quaternary complex with a cyclin, a CDK and the proliferating cell nuclear antigen (PCNA), one of the DNA replication and repair factors, suggesting that this complex might play an important role in maintaining the integrity of the genome. Here, we have focused on the p21-PCNA interaction in the context of DNA replication or DNA repair, presenting the data from both in vitro and in vivo studies of the p21 function.

C1-C2 point monitoring of low-dose cyclosporin a given as a single daily dose in children with steroid-dependent relapsing nephrotic syndrome.

AIM: It has been reported that the pharmacokinetics of cyclosporin A (CsA) in children is different from that in adults. It appears that, in general, pediatric patients metabolize CsA more rapidly than adult patients, necessitating the use of higher dose of the drug in pediatric transplant recipients. In this context, we speculated that single-dose daily administration of low-dose CsA may be associated with a higher peak blood level without associated trough blood level elevation, and thereby yield better results and allow safer use of the drug than the conventional twice daily administration dosing used for the treatment of childhood idiopathic nephrotic syndrome (INS). METHODS: A total of 10 children with steroid-dependent relapsing INS (9 with biopsy-proven minimal-change disease) who showed steroid toxicity were enrolled in the study. The initial daily dose of CsA (Neoral) used was around 2.0 mg/kg, given as a single daily dose before breakfast. The dose was subsequently adjusted to achieve a C1-C2 point blood level between 600 - 800 ng/ml. The dose of the concomitantly administered prednisolone was tapered following the commencement of CsA. RESULTS: The mean daily CsA dosage, the mean C1-C2 point blood level and the mean trough blood level in the subjects were 2.2 +/- 0.8 mg/kg, 754.0 +/- 71.9 ng/ml and 42.7 +/- 29.2 ng/ml, respectively. At the latest observation, after a mean duration of 17 months (6 - 24 months) of CsA therapy, the minimum dose of prednisolone required for maintenance of clinical remission and the calculated relapse rate were significantly decreased as compared to the respective pretreatment values (0.52 +/- 0.46 mg/kg on alternate days, vs. 0.97 +/- 0.63 mg/kg on alternate days, and 0.28 +/- 0.32 times per six months, vs. 1.06 +/- 0.41 times per six months, respectively, p = 0.005). No significant change was observed in the mean estimated GFR value as compared to the pretreatment value (183.1 +/- 35.4 ml/min/1.73 m2vs. 185.4 +/- 39.3 ml/min/1.73 m2). No evidence of CsA nephrotoxicity was observed in a repeat renal biopsy performed around 12 months after the commencement of CsA therapy in two patients. CONCLUSIONS: Despite the limitations of the study, our results suggest that administration of low-dose CsA as a single daily dose with C1-C2 point blood level monitoring might be an equally effective and safe and, therefore, more cost-beneficial, protocol for the treatment of steroid-dependent cases of relapsing INS, as conventional twice-daily administration of CsA with trough blood level monitoring. Further studies to confirm the long-term efficacy and safety of this CsA treatment protocol in larger numbers of patients are, however, needed.

Craniopharyngioma with midbrain involvement.

An adult patient had a cranlopharyngloma that involved and displaced the midportion of the midbrain, producing Weber's syndrome. Oculomotor paresis in cranlopharyngloma is not always due to parasellar involvement but may be due to midbrain involvement.

Reversibility of Parinaud syndrome in thalamic hemorrhage.

Parinaud syndrome, associated with a left thalamic hemorrhage, disappeared completely in a 57-year-old woman after ventriculoperitoneal shunt. The syndrome may be attributed to increased intracranial pressure owing to mass effect on the pretectal region and tectum, or to tightness in the incisura causing hydrocephalus secondary to aqueduct compression. The lesions responsible for the syndrome may not be irreversible.

Survival-promoting activity of IL-7 on IL-2-dependent cytotoxic T lymphocyte clones: resultant induction of G1 arrest.

Interleukin-7 (IL-7) is essential for both T cell and B cell development. Recent studies have suggested that IL-7 also functions as a survival-promoting factor for resting and activated T cells. In this study we examined the effects of IL-7 on survival and cytotoxicity of tumor-specific CD8(+) cytotoxic T lymphocyte (CTL) clones established and maintained either with IL-2 alone or with a combination of IL-2 and IL-7. While the CTL clones cultured in IL-2 alone died around day 10, the CTL clones cultured in the presence of IL-2 and IL-7 survived for more than 4 weeks after seeding. The long-term survival of the latter was correlated with the presence of IL-7 in the medium. In addition, IL-7 alone prolonged survival of other IL-2-dependent CTL clones after the removal of IL-2. IL-7 maintained the CTLs in G1 arrest after a slight proliferation during the initial phase during which low-level but sustained DNA synthesis was observed. However, there was no direct correlation between DNA synthesis and enhancement of long-term survival by IL-7 as demonstrated by the inhibiting proliferation of the CTL clones with the protein kinase inhibitor genistein. During long-term survival in the presence of IL-7, the cytotoxic activities of the CTL clones decreased gradually to background levels although they were restored soon after the next passage. These results suggested that IL-7 had the ability to set machinery in motion against apoptosis in the IL-2-dependent CTL clones. Such an effect of IL-7 might play a role in vivo in the process leading activated T cells to the resting, that is, memory state.

Role of endothelium in regulation of smooth muscle membrane potential and tone in the rabbit middle cerebral artery.

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA). 2. In endothelium-intact strips, histamine (His, 3-10 microM) and high K+ (20-80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 microM), 5-hydroxytryptamine (10 microM) and 9,11-epithio-11, 12-methano-thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+-induced contraction). 3. N(G)-nitro-L-arginine (L-NOARG, 100 microM), but not indomethacin (10 microM), greatly enhanced the phasic and the tonic contractions induced by His (1-10 microM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3-3 microM) caused concentration-dependent relaxation (maximum relaxation by 89.7 +/- 7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 microM), but not glibenclamide (3 microM), abolished the relaxation induced by ACh (0.3-3 microM) in L-NOARG-treated strips (n=4, P<0.05). 4. In endothelium-intact tissues, His (3 microM) depolarized the smooth muscle membrane potential (by 4.4 +/- 1.8 mV, n = 12, P < 0.05) whereas ACh (3 microM) caused membrane hyperpolarization (-20.9 +/- 3.0 mV, n = 25, P< 0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (-23.5 +/- 5.9 mV, n=8, P<0.05) or glibenclamide (-20.6 +/- 5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to - 5.8 +/- 3.2 mV, n = 3, P< 0.05). 5. Sodium nitroprusside (0.1-10 microM) did not hyperpolarize the smooth muscle cell membrane potential (0.2 +/- 0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n-4, P<0.05). 6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.

Neuroprotection by glial cells through adult T cell leukemia-derived factor/human thioredoxin (ADF/TRX).

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin (TRX) with many biological functions and is induced by various stimuli and stress. In the central nervous system (CNS), expression of ADF/TRX occurs in glial cells during ischemia and reperfusion. We showed that ADF/TRX was actively released from U251 astrocytoma cells upon exposure to a low concentration of H2O2. The addition of conditioned medium from H2O2-stimulated U251 cells or recombinant ADF (rADF) to the culture medium promoted the survival of neurons from embryonic mouse cortex and striatum, but the addition of mutant ADF (mADF), which has no reducing activity, did not. In addition to rADF, incubation with two other thiol compounds, 2-mercaptoethanol (2-ME) and N-acetyl-L-cysteine (NAC), also increased the neuronal cell survival rate. In contrast, L-buthionine-(S,R)-sulfoximine (BSO), which inhibited the synthesis of glutathione (GSH), decreased the neuronal cell survival rate. Intracellular GSH was increased by incubation with rADF for 24 h, as it is with 2-ME and NAC. Redox active molecules such as thiol compounds may be survival factors for central neurons in vitro, and this capacity may be supplied by endogenous molecules, such as ADF/TRX and glutathione, under certain pathologic conditions in vivo.

Bilateral intranigral NMDA infusion suppresses neuronal injury without affecting the duration of kainic acid-induced seizures in rats.

The substantia nigra pars reticulata (SNpr) is recognized as an important modulator of seizures within the limbic system. We have investigated the effects of N-methyl-D-aspartate (NMDA) infusion into SNpr upon seizure-related neuronal injury (assessed by expression of the 72-kDa heat shock protein - HSP 72) induced by systemic kainic acid (KA) in rats. Three to four days following implantation of guide cannulae for drug administration into SNpr, KA (7 mg/kg) was injected intravenously to induce seizures. Bilateral intranigral infusion of NMDA (20 nmol) 15 min prior to KA injection, suppressed the expression of HSP 72 in the hippocampal CA1 region without affecting seizure duration. These results support the involvement of NMDA receptors within SNpr in modulating neuronal injury following KA-induced limbic seizures.

Fracture of the sella turcica.

We report the cases of three patients with fracture of the sella turcica. All three patients developed vascular complications. Patient 1 had a traumatic aneurysm, Patient 2 had stenosis of the intracranial internal carotid artery, and Patient 3 had bilateral carotid-cavernous fistulas. We emphasize that, when fracture of the sella turcica is demonstrated, cerebral angiography is still the method of choice for the detection of intracranial vascular injury.

Intracerebral hemorrhage remote from the site of the initial neurosurgical procedure.

Four cases of intracerebral hemorrhage remote from the site of the initial neurosurgical procedure are presented. Two of the four patients had preoperative hypertension. Possible mechanisms are discussed, and labile hypertension and unstable blood pressure during the perisurgical period may be contributory. Clinical awareness of this rare but potential complication is essential to early diagnosis and treatment. Difficulty in awakening from anesthesia and the development of new neurological deficits not attributable to the operative site are the most important keys to early diagnosis. Computed tomography is the diagnostic method of choice.

Chondroma of the spine in a newborn infant: case report.

We report a newborn infant with a chondroma of the spine covered by a large lipoma. The child had no neurological deficit. Presenting as a tumor of the back, the chondroma was successfully excised. A review of the pertinent literature emphasizes the rarity of this lesion.

Saccular aneurysm associated with absence of the left cervical carotid arteries.

The authors report successful surgical treatment of a patient with an anterior communicating artery aneurysm associated with absence of all of the left cervical carotid arteries. A review of the previous reports of 15 cases of intracranial aneurysm in association with absence of the internal carotid artery emphasizes the rarity of this lesion. Aneurysms with such an association have a distinct distribution different from that observed in the usual population. The data suggest that intracranial aneurysm develops more frequently in the absence of the internal carotid artery as a result of altered hemodynamics.

Transvenous balloon occlusion of the cavernous sinus: an alternative therapeutic choice for recurrent traumatic carotid-cavernous fistulas.

We report a case of traumatic carotid-cavernous fistula (CCF) that recurred some 9 years after carotid trapping. The recurrent CCF was accompanied by a huge aneurysmal dilatation of the cavernous sinus. Transarterial balloon occlusion of the proximal internal carotid artery failed to occlude the fistula completely because of collateral flow to the fistula. the fistula was completely occluded by a transvenous approach via the jugular vein and inferior petrosal sinus using detachable balloons. The transjugular-inferior petrosal approach to the cavernous sinus can be an alternative for the treatment of traumatic CCF when the transarterial approach has failed to occlude the cavernous sinus.

Surgical treatment of ossification of the posterior longitudinal ligament in the thoracic spine.

Thoracic ossification of the posterior longitudinal ligament (OPLL) is a rare entity causing thoracic myelopathy. Its surgical decompression is still challenging. Three patients admitted with progressive myelopathy due to thoracic OPLL are described. A transthoracic anterolateral approach was used in the first and second cases, in which OPLL was located at the T3-T4 and T5-T6 and at the T7-T8 levels, respectively. In the third case, a transsternal approach was adopted for OPLL at the T1-T2 level. The OPLL, including dural ossification, was removed by microsurgical techniques as extensively as possible. Myelopathy in all three cases became relieved or stable postoperatively. Operative procedures are described in detail. From the viewpoint of surgical anatomy, the selection of operative approach depends on the level of the OPLL. The authors emphasize that a transthoracic anterolateral approach is the treatment of choice for extensive anterior pathology such as OPLL involving more than two thoracic bodies below the T4. A transsternal approach can provide excellent access to a lesion at the upper three thoracic bodies.

Successful removal of air gun bullets from the third ventricle.

A patient with air gun bullets in the 3rd ventricle associated with delayed ventricular hemorrhage is presented. Through an anterior transcallosal approach, the surgeon successfully removed the bullets without any significant permanent sequelae.