Retinal functional change caused by adenoviral vector-mediated transfection of LacZ gene.

We examined the effect of insertion of an exogenous gene on retinal function to assess the rationale of adenoviral vector-mediated gene transfer for future gene therapy. An adenoviral vector expressing bacterial LacZ (AdCALacZ) was injected into the eyes of adult rats either intravitreally (group A) or subretinally (group B), and the gene expression and retinal function were thus examined at different time points after gene transfer for 3 weeks. X-Gal histostaining showed that neural retinal cells were transfected in group A and that retinal pigment epithelial cells were transfected in group B. The gene transfer was more efficient in group B (54.4% of the fixed retinal area was stained) than in group A (10.4%). The electroretinogram (ERG) revealed retinal dysfunction in the AdCALacZ-transfected rats even at the stage in which the histological damage was not apparent by electron microscopy and immunohistochemical studies for cytokeratin, S-100 protein, and glial fibrillary acidic protein. The ERG change was correlated with the intensity of inflammation, and retinal function recovered to the original level by 3 weeks, along with a diminution of inflammation. Functional changes were more evident in eyes treated with AdCALacZ than in those infected with adenoviral vector with no exogenous gene; however, no histological difference was observed between these groups, indicating that the insertion of exogenous gene itself affects retinal function. The results showed that different kinds of retinal cells could be gene-transferred by an adenoviral vector, depending on the application method. The retinal dysfunction caused by each adenoviral transfection method was caused by inflammation and the insertion of exogenous gene, and this retinal dysfunction was recoverable. In future gene therapy, special attention should be given to the method of exogenous gene insertion in the retina.

Changes in the phosphorylation states of connexin43 in myoepithelial cells of lactating rat mammary glands.

Using specific antibodies and cDNA probe, we examined the expression pattern of a major gap junction protein, connexin43 (Cx43), in rat mammary glands during pregnancy and lactation. Double immuno-fluorescence revealed that the labeling of Cx43 was superimposed in the alpha-smooth muscle actin-positive cells, suggesting that myoepithelial cell were interconnected by gap junctions formed of Cx43. Just after delivery, the Cx43-labeled plaques were enlarged and increased in intensity. Northern and Western blot analyses confirmed the dramatic induction of Cx43 at both mRNA and protein levels on the day of parturition. Cx43 mRNA transcript immediately declined, while the increase of Cx43 protein continued for a few days. During pregnancy, immunoblots showed two bands of almost equal amounts at 43 and 45 kDa. Following delivery, the 45-kDa band gradually increased in intensity with a concomitant decrease of the 43-kDa band. From the sixth day of lactation, Cx43 was always detected as a single band at 45 kDa. Alkaline phosphatase treatment of immunoprecipitated Cx43 revealed that both bands represented phosphorylated forms, thus indicating that Cx43 was naturally phosphorylated and that it altered its phosphorylation states during lactation stages. These results suggest that the induction of Cx43 with the changes in the phosphorylation states plays an important role in the lactating function of myoepithelial cells in rat mammary glands. This is the first report on the changes of Cx43 phosphorylation states during physiological stages in vivo.

Microglial localization of alpha-interferon receptor in human brain tissues.

The localization of the alpha-interferon receptor (alpha-IFNR) was examined in human brain tissues from neurologically normal, Alzheimer's disease (AD) and cerebral infarction cases, using one antibody (IFNaR3) which preferentially recognizes alpha 2-IFN receptor and a second antibody (anti-p40) which recognizes the soluble alpha-IFN alpha/beta receptor. In all cases, microglial cells were stained with the antibodies. In infarct brains, macrophages were intensely labeled for alpha-IFNR. The anti-p40 antibody also showed capillary staining which might be due to residual blood plasma. On Western blots, IFNaR3 recognized only a 210 kDa alpha-IFN/receptor complex, indicating its localization in microglial cells. The anti-p40 antibody recognized this complex as well as four other major materials of lower molecular weight. These results suggest that alpha-IFNR protein is constitutively expressed in microglial cells.

Specific detection of circulating tumor cells by reverse transcriptase-polymerase chain reaction of a beta-casein-like protein, preferentially expressed in malignant neoplasms.

Beta-casein-like protein (BCLP) is a putative protein on cervical cancer and exhibits immunological characteristics similar to those of bovine beta-casein. We evaluated if BCLP mRNA detection in the blood is useful in gynecologic malignancies. We examined 30 patients with uterine cancer, nine cultured cancer cell lines and 26 healthy women volunteers. From these study populations and samples, total RNA was obtained. Reverse transcriptase-PCR (RT-PCR) of BCLP was performed on each sample. Eighteen (60.0%) patients and 4 (15.4%) volunteers were positive for BCLP mRNA. The RT-PCR reached sensitivity and specificity of 60.0% and 84.6%, respectively. Of eight patients having diagnosed recurrence, 7 (87.5%) were positive for BCLP mRNA. In patients with recurrence, sensitivity and specificity were 87.5% and 50%, respectively. The expression of mRNA showed a correlation with recurrence, but no correlation with metastasis or histological type. BCLP was specifically expressed in cancer cells and might be an aid in clinical diagnosis.

Invasion of brain by neurovirulent influenza A virus after intranasal inoculation.

We examined the immunohistochemical localization of neurovirulent influenza A virus (WSN and R404BP) in the brains and lungs of C3H/HeN and A2G mice 3 days after intranasal inoculation, as well as in A2G mice 3 days after intracerebral inoculation. The invasion of brain by R404BP virus was greater than that of WSN in both mouse strains. The areas of infection were less extensive in Mx gene(+) A2G mice than in the Mx gene(-) C3H/HeN mice, which is consistent with previous reports. Major involvement was seen in various catecholaminergic neurons, capillaries, meninges and ependymal areas. Olfactory and trigeminal nerves were not positive for the WSN antigen. These results show that, in the early phase of infection, influenza A virus invades, probably through hematogenous spread, and particularly affects catecholaminergic neurons.

Leech extracellular hemoglobin: two globin strains that are akin to vertebrate hemoglobin alpha and beta chains.

Leech (Whitmania edentula, Haemadipsa zeylanica var. japonica and Erpobdella lineata) extracellular hemoglobins are basically composed of three constituent subunits, a dimer (D1 and D2 chains) and two monomers (M1 and M2 chains). We isolated these four chains from respective species by a combination of reversed-phase chromatography on a Resource RPC column and gel-filtration on a Superdex 75 column. The apparent molecular masses of the four globin chains were estimated by SDS-PAGE analysis to be 13 kDa (M1), 16 kDa (M2; 19 kDa in its reduced form) and about 27 kDa for the dimer subunit (13 kDa for D1; 15 kDa for D2), regardless of the source. The amino (N)-terminal segments (21-30 residues) from twelve globin chains of the above three species were determined and aligned. It was found that the twelve sequences could be separated into two distinct globin groups A and B. This finding supports the original idea of "two globin strains in annelid hemoglobin", which was proposed without any evidence for leech hemoglobins. Comparing the sequences in the three classes of Annelida, Hirudinea, Oligochaeta and Polychaeta, we found two invariant amino acids, Cys and Trp, which are interposed by eleven amino acid residues. Furthermore, the globin chains belonging to strain A were readily discernible as they had three more invariants, Ser-13, Asp-16 and Trp-28, while the globin chains of strain B had two more invariants, Lys-12 and Arg-27. Consequently, we propose that each of the three classes of Annelida have two distinct groups of globin chains that are akin to vertebrate hemoglobin alpha and beta chains.

Complete hydatidiform mole coexisting with a live fetus after gamete intrafallopian transfer. A case report.

BACKGROUND: Molar pregnancy with a coexisting live fetus is a rare occurrence. We report the only known case with a surviving coexistent fetus after gamete intrafallopian transfer (GIFT). CASE: After GIFT, a 28-year-old primary infertility patient was diagnosed as having a complete hydatidiform mole coexisting with a live fetus at 13 weeks of gestation. At 36 weeks of gestation, a cesarean section was performed due to elevated serum human chorionic gonadotropin (hCG) levels, and a male infant with a normal appearance and weighing 2,688 g was delivered. CONCLUSION: If the patient desires to try to carry the fetus to viability after counseling on the possible associated risks of malignancy, it is possible to achieve fetal viability if (1) there is decline in the serum hCG level after it peaks before the second trimester, (2) ultrasound reveals degeneration of the molar part, and (3) there are no complications of pregnancy.

Increased expression of angiotensin-converting enzyme in retinas of diabetic rats.

PURPOSE: The aim of this study was to examine the localization and the changes in the amount of angiotensin-converting enzyme (ACE) and the relationship between the renin-angiotensin (RA) system and vascular endothelial growth factor (VEGF)/VEGF-receptor system in the retinas of diabetic rats. METHODS: Immunohistochemical localization of ACE, VEGF, and VEGF-receptor fetal liver kinase-1 (Flk-1) was examined in cryosections of the retinas of streptozotocin-injected diabetic rats. A semi-quantitative comparison of diabetic rats with age-matched controls was also performed by counting the ACE- or Flk-1-positive vessels per microscopic field. RESULTS: ACE immunoreactivity was localized in the retinal vessel walls, and the percentages of ACE-positive vessels were significantly increased in the retinas of diabetic rats maintained 3 to 5 months. Both VEGF and Flk-1 signals increased simultaneously with the increment of ACE immunoreactivity. CONCLUSIONS: ACE, expressed in the retinal vessel walls, increases simultaneously with the increment of both VEGF and Flk-1 in the retinas of diabetic rats, suggesting that upregulation of ACE might play some role in the progression of diabetic retinopathy through the VEGF/VEGF receptor system.

[A case of sepsis by Neisseria meningitidis beginning with pneumonia during a trip abroad].

A 62-year-male presented a high fever and a dry cough during a trip to Australia. He was admitted to a hospital as soon as be returned to Japan. The next day after returning to Japan, he was transferred to our hospital with septic shock and loss of consciousness. Neisseria meningitidis was cultured from his blood. N. meningitidis is rare in Japan. However its seems common, in some foreign countries. With these findings, it can be postulated that N. meningitidis might be one of the etiological agents of the imported infectious disease.

[Asynergia in cerebellar ataxia--the significance of impairment of elbow fixation in forearm pronation-supination test].

Patients with cerebellar ataxia often show swing of the upper arms or the elbows in forearm pronation-supination test (FPS test). Eleven healthy volunteers and sixteen patients with cerebellar ataxia were examined. We observed FPS test of the subject with their shoulder and elbow joints being flexed at a right angle respectively in sitting position, and recorded them by VTR. We noticed following four points in FPS movement: 1) velocity and regularity, 2) amplitude, 3) finger posture, 4) state of elbow fixation. We recorded surface EMG of the upper arm-fixing muscles (deltoid, pectoralis major, infraspinatus) and the forearm-moving muscles (biceps brachii, pronator teres). In controls, FPS movements were regular and speedy. The upper arm was so well fixed that the elbow did not swing. Continuous discharges were recorded from upper arm-fixing muscles when the upper arm was lifting. Reciprocal grouping discharges were recorded during FPS movements from upper arm-fixing muscles so that they were suppressing the swing of the elbow. In ataxic patients, FPS movements were dysrhythmic and slow. The elbow swung during FPS test. We observed internal rotation and abduction of the shoulder when forearm pronated, and outer rotation and adduction of the shoulder when forearm supinated. Reciprocal grouping discharges that were reverse of normal ones were recorded from upper arm-fixing muscles so that they were aggravating the swing of the elbow. Synergia is an ability to accomplish the various muscle contractions coordinately when making up a certain action. Asynergia is one of the characteristic features of disturbance of the association of muscle contractions of the upper arm and the forearm. FPS test is easy and useful to detect asynergia of the upper extremity in cerebellar ataxia.

["Acro-erythro-cyanosis"--peculiar vasomotor symptoms due to cervical hernial myelopathy].

Two cases of cervical myelopathy, which exhibited peculiar vasomotor symptoms ("acro-erythro-cyanosis") on distal regions of the four limbs are reported. Continuous reddening, swelling and skin temperature increase were observed on both hands and feet in case 1, a-44-year-old man, and on both hands in case 2, a-47-year-old man. Cold stimulation resulted in cyanosis and decrease of skin temperature on the affected regions paroxysmally. The condition of skin at room temperature may be caused by arteriectasia of arterioles due to hypotonia of vasomotor fibers, and this was similar to erythromelalgia. On the other hand, cold stimulation may have led to this condition, where the contraction of skin arterioles due to hypertonia of vasomotor fibers was added to the dilation of venule, and this was similar to acrocyanosis. Consequently, we provisionally named the vasomotor symptoms in the present cases as "acro-erythro-cyanosis". The other neurological signs were as follows. Case 1: mild weakness in right upper and lower limbs and left small hand muscles, mild superficial and deep sensory disturbance on bilateral palms and soles and decrease of vibration on bilateral lumbar regions and thereunder. Case 2: mild weakness of right small hand muscles, superficial sensory disturbance on distal regions of bilateral upper and lower limbs and a decrease of joint position sense on right hand. Myelography and metrizamide CT myelography revealed a high-degree deformity of the spinal cord due to the herniated disks between C4 and C5 in case 1 and between C3 and C4 in case 2.(ABSTRACT TRUNCATED AT 250 WORDS)

[Type and screen system for efficient use of blood on elective surgery].

For the efficient use of blood resources, we have adopted the Type and Screen system (T & S) and Maximum Surgical Blood Order Schedule (MSBOS) for elective operations. To evaluate the usefulness of these systems, we analyzed the crossmatched to transfused blood ratio (C/T ratio) for the period of one year before and after the adoption of the systems. C/T ratio were 4.65 and 2.38 respectively and crossmatched blood units decreased from 3799 to 2511 units. These results show the usefulness of these systems in perioperative blood transfusion system. It is possible to make use of blood more efficiently by further detailed re-evaluation of both T & S and MSBOS systems.

[Anesthetic management in a patient with severe acute pancreatitis during pregnancy].

Continuous epidural anesthesia was used in a 34 year-old pregnant woman with acute pancreatitis related to hypertriglyceridemia. She underwent an emergency cesarean section due to severe pancreatitis under spinal anesthesia. After delivery, extended incision was made to examine the pancreas and to perform drainage. Epidural infusion using 1% mepivacaine and buprenorphine was started to reduce pain and improve microcirculation. After starting epidural infusion with other therapies, clinical feature and data improved. This case suggests that reduction of severe pain and improvement of microcirculation are important in therapies of severe pancreatitis.

[Right phrenic nerve palsy in a patient with ventricular septal perforation after acute myocardial infarction].

A 77-year-old female had acute myocardial infarction and ventricular septal perforation (VSP). An operation was performed 14 days after VSP. During cardiopulmonary bypass, a patch was sutured in place on the left side of the defect under a mild hypothermia with topical cardiac cooling procedure. In the postoperative period, mechanical ventilation was continued with use of pulmonary monitor (CP-100, BICORE). When the patient was weaned of mechanical ventilation, work of breathing increased (WOBp: 1.67 J.l-1) without the change of SaO2, PaO2, PaCO2 and the respiratory state. After extubation, the chest X-ray showed elevated right diaphragmatic level and a diagnosis of unilateral diaphragmatic paralysis was made. Oral intake was started two days after extubation. SpO2 decreased after oral intake, and it was significantly improved by taking a left lateral position. The symptoms disappeared 47 days after the operation. It should be noticed that the rise of endoceliac pressure in the patient who had a unilateral phrenic nerve palsy, affected the SpO2.

[Severe theophylline toxicity in a pregnant asthmatic patient].

A 32 year old pregnant woman who had taken large doses of theophylline for asthmatic attack was brought to our emergency medical center with convulsion and respiratory disturbance. Serum theophylline concentration was 52 micrograms.dl-1 on admission. We intubated the patient to control convulsion and respiration and performed cesarean section. Serum theophylline concentration of the newborn was almost at the same level as that of her mother. Baby was placed on ventilator for several days and discharged without trouble. We must consider the pregnancy as a major risk factor of theophylline toxicity.

[Anesthetic management for a patient with autoimmune hemolytic anemia].

A 69-year-old woman with autoimmune hemolytic anemia (AIHA) received gastric resection and splenectomy under general anesthesia combined with epidural anesthesia. Her blood type had been determined as type A, Rh type CCDee, with anti-e-antibody and anti-pdl-antibody positive as autoantibodies. We applied the technique of hemodilutional autologous transfusion to supplement the blood loss during the operation to prevent hemolysis or occurrence of new antibody after homologous transfusion. We should pay much attention to a patient with AIHA to find signs of peri-operative hemolysis and to give the treatment for hemolysis in early stage. We consider that the hemodilutional autologous transfusion is a useful technique for the anesthetic management of the patient having a rare blood type.

[A case report: partial anomalous pulmonary venous return of the left lung communicating with the left atrium through the aberrant vein].

A rare case with partial anomalous pulmonary venous return was presented. The left superior pulmonary vein was drained into the left innominate vein via a common vertical vein and it also communicated with the left atrium via a small aberrant vein. All the other pulmonary veins were normally drained into the left atrium. The surgical repair was performed through the midline sternotomy. The junction of the vertical vein and the left innominate vein was ligated with slight elevation of the pressure in the left pulmonary vein. Under the cardiopulmonary bypass, the secundum atrial septal defect was closed by direct sutures. The postoperative course was uneventful and there was no pulmonary congestion in the left upper lung field.

[A case of lateral origin of the left internal mammary artery].

A 66 years old men, with left anterior descending coronary artery (LAD) stenosis and aortic valve stenosis, underwent coronary artery bypass grafting (CABG) to LAD with left internal mammary artery (LIMA) and aortic valve replacement. His postoperative course was uneventful. But, postoperative angiogram showed that his patent LIMA graft was originated from much lateral side of the left subclavian artery. Internal mammary artery is considered the most ideal graft for CABG, but its' anomality is not well known. We present this rare case with the anomalous origin of LIMA.