Expression of vascular endothelial growth factor receptors in bovine cystic follicles.

Cystic follicles have excess fluid derived from blood flow in the theca interna of the follicle; therefore, the vasculature network is related to cystic follicle formation. Vascular endothelial growth factor (VEGF) is a potent stimulator of blood vessel permeability and angiogenesis. The aim of this study was to examine the expression of VEGF receptors proteins and mRNA in cystic follicles to elucidate the VEGF system in cystic follicles. The expression of protein for VEGF receptors; fms-like-tyrosine kinase-1 (Flt-1) and foetal liver kinase-1 (Flk-1) was detected by the immunohistochemical method. The mRNA expression of Flt-1 and Flk-1 in cystic follicles was determined by RT-PCR. Concentration of oestradiol-17beta and progesterone in the follicular fluid of cystic follicles was determined using ELISA. Flt-1- and Flk-1 proteins were localized in granulosa and theca interna cells and endothelial cells of theca layers. The intensity of Flt-1 and Flk-1 immunoreaction was similar among cystic follicles with various ratios of oestradiol-17beta/progesterone concentrations. The expression of Flt-1 and Flk-1 mRNA was similar, regardless of the ratio of oestradiol-17beta to progesterone in follicular fluid. These results demonstrate that cystic follicles have both VEGF receptors in the granulosa and theca interna layers, which may be responsible for the increased permeability of microvessels, causing the accumulation of follicular fluid in cystic follicles.

Microvascular distribution and vascular endothelial growth factor expression in bovine cystic follicles.

The aim of this study was to examine the distribution of microvessels in the theca and the expression of vascular endothelial growth factor (VEGF) in the theca and granulosa of cystic follicles. Paraffin sections of cystic follicles were stained with Bandeiraea simplicifolia-I (BS-I) to visualize the endothelial cells of microvessels. The other sections were immunostained with anti-VEGF antibody. The mRNA expression of VEGF in the theca interna of cystic and healthy follicle was determined by RT-PCR. In the theca interna, cystic follicles with granulosa cells had significantly greater microvessel number density (the number of microvessels per given field) and area (area occupied by microvessels per given area) than healthy follicles in various sizes (<3, 4-8, >9 mm). Loss of granulosa cells from cystic follicles resulted in a similar number density, but significantly smaller area of microvessels in the theca interna. There was no significant difference in the microvessel number density and area of the theca externa between the types of follicle. VEGF protein was expressed in the granulosa and theca interna of healthy and cystic follicles. These results demonstrate that cystic follicles have a highly developed vasculature network in the theca interna, especially in cystic follicles containing granulosa cells. It is also suggested that VEGF is highly expressed in the cystic follicle as well as healthy follicle, which may be associated with advanced vasculature and the accumulation of follicular fluid in cystic follicles.

Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme.

DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus sp. KOD1) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, when long distance PCR (>5 kbp) was performed with KOD DNA polymerase, amplification efficiency (product yield) becomes lower because of its strong 3'-5' exonuclease activity for proof-reading. In order to improve a target length limitation in PCR, mutant DNA polymerases with decreased 3'-5' exonuclease activity were designed by substituting amino acid residues in conserved exonuclease motifs, Exo I (Asp141-Xaa-Glu), Exo II (Asn210-Xaa-Xaa-Xaa-Phe-Asp), and Exo III (Tyr311-Xaa-Xaa-Xaa-Asp). Exonuclease activity and amplification fidelity (error rate) of the DNA polymerases were altered by mutagenesis. However, long and accurate PCR by a single-type of mutant DNA polymerase was very difficult. The wild-type DNA polymerase (WT) and its exonuclease deficient mutant (N210D) were mixed in different ratio and their characteristics in PCR were examined. When the mixed enzyme (WT and N210D) was made at the ratio of 1:40, long PCR (15 kbp) at lower mutation frequency could be efficiently achieved.

Analysis of the proliferative potential of tumor cells after stereotactic radiosurgery for recurrent astrocytic tumors.

We analyzed the effectiveness of stereotactic radiosurgery (SRS) for recurrent astrocytic tumors histologically. Five patients were followed by pathological examination after radiosurgery treatment of recurrent astrocytic tumors. Histological diagnoses at the time of the last operation before SRS were Daumas-Duport grade II in two patients and grade IV (glioblastoma) in three patients. No histological diagnoses at the time of SRS were identified in any patients. Contrast enhanced lesions enlarged gradually on magnetic resonance (MR) images after SRS, and local control by SRS was judged as progressive disease radiologically in all patients. Four of five patients received re-operation after SRS, and the other patient died without re-operation and underwent post-mortem examination. After SRS, Ki-67 labeling indices (LIs) of recurrent astrocytomas initially diagnosed as grade II were 2.6% and 1.1%. These LIs were relatively lower than those of the control group of patients with recurrent grade II astrocytomas that were not treated by SRS. Ki-67 LIs of three glioblastomas after SRS were 23.5%, 18.6%, and 17.8%. These LIs were significantly lower than those before SRS (2.3%, 4.5%, and 0.9%). In the autopsy case, there was a significant difference between the LI of tumor cells in the radiosurgically treated region (0.9%) and that in the untreated region (29.2%). These results suggest that the proliferative potential of malignant astrocytic tumors in the radiosurgically treated area is reduced after SRS, and that radiological enlargement of enhanced lesions on MR images is due to propagation of the residual tumor cells that were not covered by radiosurgical target volume or to radiation necrosis. SRS may be a useful therapeutic tool in multidisciplinary treatment of malignant gliomas.

Surgical transvenous embolization of a cortically draining carotid cavernous fistula via a vein of the sylvian fissure.

Percutaneous transvenous embolization is one of the most effective treatments of intracranial dural arteriovenous fistulas (AVFs) involving the dural sinuses. We present a unique case of surgical transvenous embolization in a 48-year-old man with a dural AVF of the cavernous sinus who presented with intracerebral hematoma. The dural AVF drained only into the vein of the sylvian fissure on angiography. Transvenous embolization via the vein of the sylvian fissure during craniotomy obliterated the AVF completely.

Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.

cDNA cloning and differential gene expression of three catalases in pumpkin.

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.

Reconstruction of the skull base using a silicone plate during transsphenoidal surgery.

Our operative experience of reconstruction of the skull base using a silicone plate during rhinoseptoplastic transsphenoidal surgery is described. A suitably shaped plate can be easily and quickly made from a large silicone block of the type that is widely used in cosmetic surgery. The plates can be distinguished on magnetic resonance images postoperatively. We consider that this technique is of use not only for repairing the sellar floor, but also for closing a wide defect of the skull base.

Brachycephalic, serrated curettes for anterior cervical spinal microsurgery. Technical note.

Modified spinal fusion curettes with two unusual features on the tip are described for anterior cervical procedures. The features are: 1) an elliptical, long sidewise ("brachycephalic") scoop at the tip and 2) a serration equipped on the distal scoop edge. These designs allow reliable manipulation and significant removal of the intervertebral space contents in the deep operative field under the microscope.

Cerebellopontine angle epidermoid tumor extending into the upper cervical spinal canal--case report.

A 61-year-old male with right facial pain and hearing loss was found to have a cerebellopontine angle epidermoid tumor that extended downward through the foramen magnum into the upper cervical spinal canal. The tumor was removed except for a small fragment of its capsule adherent to the brainstem. Only three such tumors with caudal extension have been reported.

[Traumatic arteriovenous fistula with feeders of the scalp, dura mater and pia mater: case report].

A very rare case of 41-year-old male with symptomatic dural arteriovenous fistula which was detected after 22 years from receiving head injury is reported. This AVF had arterial blood supply of scalp, dural and pial origins. In the literature, only two similar cases have so far been reported. The draining veins were the superior sagittal sinus and the vein of Trolard, and a large vascular sac was seen in the pial vein.

[Change of plasma volume on patients treated by induced hypertension therapy for vasospasm].

UNLABELLED: Plasma volume, central venous pressure and water balance of 24 patients were measured about 1st and 2nd week after early operation for the ruptured aneurysms. All of the patients had been treated by induced hypertension therapy for delayed vasospasm successfully. Plasma volume was measured by RI method using RISA. The average of plasma volume of 16 patients was 58.3 +/- 6.0 ml/kg. This group had 200 ml of 25% albumin every day. It was 48.2 +/- 6.2 ml/kg on 8 patients without albumin. The average of CVP was 11.3 +/- 2.2 cm on the albumin group. It was 5.3 +/- 1.5 cm on the other group. The value of plasma volume and CVP was higher statistically on the albumin group than the other group. Water balance was positive on about half of the albumin group. It was negative on the other group. Three patients of the albumin group had pulmonary edema. THE CONCLUSION: The state of patient's hydration is not hypovolemic, as far as the induced hypertension therapy is possible, even though patient's CVP is low and water blance is negative. Albumin lets the plasma volume increase easily. Therefore, the risk of pulmonary edema may rise when only unreliable CVP is used for the monitoring of plasma volume. Plasma volume measurement is indispensable to the monitoring of fluid management for vasospasm and plasma volume should be aimed at 55-60 ml/kg.