A standardized system for assessment and delivery of nutrition support in a large teaching hospital. The nutrition support service (NSS) is consulted to evaluate and provide nutrition support to 500 patients per year. To facilitate this process, three forms--a consultation request form and preprinted parenteral and enteral nutrition order forms are used. The NSS Consult Request form was developed to provide consistency in medical, dietetic, nursing, and pharmacy patient assessments. This form is organized so that it includes clinical information necessary for nutrition assessment. Specifically, the NSS completes a nutrition assessment that includes a diet history; indirect calorimetry, only when indicated; a laboratory (metabolic) assessment; a clinical impression used to put the above-mentioned information into a nutrition perspective; and recommendations for either enteral or parenteral therapy. The recommendation for implementing nutrition support as either total enteral nutrition or total parenteral nutrition is made by using the respective order forms for enteral or parenteral nutrition. These include orders for specific formulas and additives and orders for laboratory monitors, nursing care, and criteria for notification of the physician. These forms facilitate the accurate transcription, preparation, and delivery of NSS orders by pharmacy, dietetics, and nursing departments. This approach provides an excellent framework in which to teach dietitians, pharmacists, nurses, and physicians a method for the delivery of appropriate nutrition support and provides a database for the performance of quality assurance analysis and clinical research.


Severe hypophosphatemia in postoperative patients. Severe hypophosphatemia may develop in postoperative patients for several reasons including alcohol withdrawal, diabetic ketoacidosis, nutritional recovery (refeeding) syndrome, and severe respiratory alkalosis. Severe hypophosphatemia may result in central nervous system abnormalities, muscle weakness, and renal, hepatic, cardiac, and respiratory dysfunction. Hypophosphatemia may be prevented by close monitoring of phosphorus concentrations in serum, especially in patients predisposed to developing this problem. Proper techniques for the maintenance and repletion of phosphate for both enteral and parenteral use are described.


Bedside postpyloric placement of weighted feeding tubes. A pilot study to evaluate a technique for the bedside placement of small-bore, weighted feeding tubes past the pylorus in critically ill patients is described. The technique involves the use of a #8 or #10 French small-bore, tungsten-weighted feeding tube, specific patient positioning, and defined insertion techniques, including gastric distention with air. The tubes were placed at the bedside in the intensive care unit by the nutrition support team nurse. Successful postpyloric placement was achieved in 85 (83%) of 103 patients. Approximate time for successful insertion was 30 minutes. A follow-up study to define factors influencing success or failure of this technique is planned.


Nutrition support teams--alive, well, and still growing. Results of a 1991 A.S.P.E.N. survey. This report summarizes data collected from 1680 hospitals that responded to the American Society for Parenteral and Enteral Nutrition (A.S.P.E.N.) Nutrition Support Team Survey. The survey was conducted primarily to determine the prevalence, composition, and funding of existing and planned Nutrition Support Teams. Results tend to verify the importance of the Nutrition Support Team within the hospital's organizational structure. In addition, A.S.P.E.N. hoped to develop a national directory of Nutrition Support Teams as a byproduct of the survey. Details about the Nutrition Support Team Directory appear at the conclusion of this report.


[The viability of a learning contract].

[The viability of a learning contract]. A basic principle of teaching is that students should be actively involved in the educational process. A learning contract is not a substitute method for learning, but enables a student to have a certain amount of control over objectives, content, methods and evaluation of certain curriculum contents. The learning contract gives the student the opportunity to take responsibility for own learning and places the student in an active role with regard to learning.


[Psychiatric nursing. The ideal and the practice]. Psychiatric services at primary health care level are far from ideal. Under present conditions the PHCN is relatively uninvolved. This state of affairs will change when a new model proposed by the authors is implemented. This model involves primary prevention programmes; training of PHCNs in the classification and diagnostic criteria of psychiatric illnesses; developing skills in crisis management, therapeutic intervention and behaviour modification; knowledge of drugs and their side-effects; multi-professional team planning implemented by the PHCN; and 24-hour consultation services by specialists.


[Migraine--considerations on the achievements in medicine]. Migraine is among the most mysterious diseases. It has been known for centuries but as yet it has resisted the advances in medicine and has not revealed its aetiology, mechanism of headache and possibilities of treatment. Various theories on the pathogenesis of migraine and arguments for and against them are reviewed here. The most convincing hypothesis seems to be that which covers all the achievements in this field, that is the neuronal-vascular theory in which serotonin is given the role of the main biochemical factor. The diagnosis of migraine is easy if its history is known, but the first attack, especially if very severe, may be difficult to diagnose and should be differentiated from meningitis or intracranial haemorrhage. The modern imaging techniques confirm the development of transient ischaemia in the brain which can explain the aura and the post-attack manifestations. The treatment includes interruption of attack and prevention of further attacks. As long as the aetiology and pathogenesis of migraine remain not fully understood, the interruption of attacks seems to be the most adequate management and here new possibilities have been demonstrated connected with the discovery of serotonin receptors. Prophylactic treatment may be justified only in severe and frequent attacks and its effectiveness is temporary. In summary it may be stated that as yet only several unshakeable facts have been established in the aetiology and pathogenesis of migraine: heritability, serotonin, vascular system of the head, trigeminal nerve, cerebral centres of inexact location and factors provoking attacks. They all are forming a chain of relationships which remains in the realm of hypotheses.


Cerebrospinal fluid flow. I. Physiology of cardiac-related pulsation. Cardiac-related motion of the cerebrospinal fluid (CSF) was investigated by analysis of the velocity-dependent phase of CSF protons and flow-dependent signal enhancement in magnitude images using ECG-gated FLASH sequences. In the cerebral aqueduct, CSF flow from the third to the fourth ventricle begins 200 ms after the R-wave of the ECG and simulates an arterial pulse wave pattern. It lasts about 60% of the cardiac cycle and is followed by backflow from the fourth to the third ventricle, which is slower and shorter. In the spinal canal, oscillating caudad motion precedes flow from the third to the fourth ventricle by about 50-100 ms and is superimposed on a bulk flow, which moves simultaneously in opposite directions in separate subarachnoid channels; it is directed mainly caudally in the anterior cervical subarachnoid space.


Cerebrospinal fluid flow. II. Physiology of respiration-related pulsations. Cerebrospinal fluid (CSF) flow in the cerebral aqueduct and spinal canal was analysed using real-time magnetic resonance imaging measurement techniques. Respiration-induced rhythmic modulation of the cardiac-related oscillating CSF pulsation in the cerebral aqueduct and spinal canal was found. Deep inspiration was immediately followed by a marked increase in downward CSF flow in the cervical spinal canal, whereas a delay of about two heart beats was seen before downward flow from the third to the fourth ventricle increased. This pattern was also detected during yawning and was followed by a marked increase of blood flow in the internal jugular vein.


Cerebrospinal fluid flow. III. Pathological cerebrospinal fluid pulsations. Cardiac- and respiration-related movements of the cerebrospinal fluid (CSF) were investigated by MRI in 71 patients. In most patients with arteriosclerotic occlusive vascular disease CSF pulsations are normal. Decreased pulsatile flow is detectable in those with arteriovenous malformations, intracranial air and following lumbar puncture and withdrawal of CSF. Increased pulsatile flow in the cerebral aqueduct was found in 2 patients with large aneurysms, idiopathic communicating syringomyelia and in most cases of normal pressure hydrocephalus (NPH). CSF flow in the cervical spinal canal is, however, reduced or normal in NPH, indicating reduction of the unfolding ability of the surface of the brain and/or inhibition of rapid CSF movements in the subarachnoid space over its convexity.


MRI of anterior spinal artery syndrome of the cervical spinal cord. Cervical spinal cord lesions in the anterior spinal artery syndrome were delineated on magnetic resonance images (MRI) in four patients. The lesion was always seen anteriorly in the cervical cord. On T2-weighted images, the lesions appeared hyperintense relative to the normal spinal cord, while on T1-weighted images, two chronic lesions appeared hypointense, with local atrophy of the cord. In one case, repeated T1-weighted images showed no signal abnormality 4 days after the ictus, but the lesion became hypointense 18 days later, when contrast enhancement was also recognized after injection of Gd-DTPA; this sequence of intensity changes was similar to that of cerebral infarction. The extent of the lesion seen MRI correlated closely with neurological findings in all cases. Although the findings may not be specific, MRI is now the modality of choice for confirming the diagnosis in patients suspected of having an anterior spinal artery syndrome.


MRI in chronic spinal cord trauma.

MRI in chronic spinal cord trauma. Eighty-seven patients aged 16-68 years have been examined by magnetic resonance imaging (MRI) following spinal injury. The MRI findings have been correlated with length of history between trauma and investigation, extent of residual function and site of injury. They include changes at the site of injury consistent with myelomalacia in 37%, a syrinx in 40%, persistent cord compression in 32% and atrophy in 18%. An extensive syrinx can develop within 2 months of injury and it is nearly twice as common in patients with complete paralysis as in those whose paralysis was incomplete. It is suggested that investigation and management of spinal trauma should include early and repeated MRI examinations to detect sequelae at an early stage.


Magnetic resonance angiography of extracranial carotid and vertebral arteries, including their origins: comparison with digital subtraction angiography. Although carotid bifurcation stenoses are not the only lesions of the extracranial cerebral arteries, magnetic resonance angiographic (MRA) studies to date have concentrated on the carotid bifurcation. We compared digital subtraction angiography of the extracranial portions of the cerebral arteries with MRA using an ordinary body coil, the time-of-flight method, and multiple transverse slabs which covered the arteries down to the aortic arch. Twenty-two patients (15 with arteriosclerotic diseases, 4 with aortitis, and 3 with tumours) had MRA using a 1.5 T magnet system with a three-dimensional fast imaging with steady state precession (FISP) technique. Thirty-nine carotid and 39 vertebral arteries were assessed by three radiologists with regard to stenoses or occlusions, graded as normal, mild (< 30%), moderate (30-60%) or severe (> 60%) stenosis, or occluded. Grading corresponded well in 81%; stenoses appeared more marked on MRA in 14% and were seen less clearly on MRA in 5%. When 26 carotid bifurcations were assessed separately, grading corresponded well in 95%. MRA is the only method which can display the whole course of the extracranial carotid and vertebral arteries non-invasively and satisfactorily.


Magnetic resonance angiography compared to intra-arterial digital subtraction angiography in patients with subarachnoid haemorrhage. In order to evaluate the sensitivity and specificity of magnetic resonance angiography (MRA) in spontaneous subarachnoid haemorrhage, 14 patients with recent haemorrhage verified by CT or lumbar puncture were investigated with both selective intra-arterial digital subtraction angiography (IA-DSA) and MRA by two independent teams, each having the same preangiographic information. The results were compared with each other and whenever possible (all positive cases except one) with those of surgical intervention. Seven patients were identified by MRA and IA-DSA as having a single aneurysm on the circle of Willis, 1 an aneurysm of the posterior inferior cerebellar artery 1 an aneurysm of the internal carotid artery (siphon) and 2 patients with two aneurysms on the circle of Willis. MRA and IA-DSA both failed to demonstrate aneurysms in 2 cases. Three patients had negative results on both methods and no surgical intervention was attempted. The aneurysms ranged from 0.3 to 1.5 cm in size. In most cases there was agreement between MRA and DSA, leading us to believe that, if the proper protocols are followed, MRA is a powerful alternative to other established methods in the detection of intracranial aneurysms. At this stage it will not replace IA-DSA prior to surgery, but the ability to obtain various projections using 3D MRA may improve surgical planning.


Magnetic resonance angiography of intracranial aneurysms: comparison with intra-arterial digital subtraction angiography. Magnetic resonance angiography (MRA) with flow rephased gradient-echo sequences is a new non-invasive method for vascular imaging. We compared MRA and intra-arterial digital subtraction angiography in 18 patients with intracranial aneurysms to test whether MRA presently provides an alternative to cerebral angiography for the diagnosis of these anomalies. MRA showed 19 of the 22 aneurysms detected (86.4%). However, problems, especially with turbulent or slow flow, resulted in 6 studies (27.3%) with limited and 2 with questionable demonstration of an aneurysm, and 1 false negative study. At present, MRA is definitely inferior to angiography for the demonstration of intracranial aneurysms, due to its lower resolution and other limitations.


Investigation of stroke in sickle cell disease by 1H nuclear magnetic resonance spectroscopy. Localized proton nuclear magnetic resonance spectroscopy (MRS), obtained with stimulated echo and spin echo sequences, MR imaging (MRI) and MR angiography (MRA) were used to study the brain in 13 children and adolescents with sickle cell disease. Regions of interest (ROI) studied by MRS included regions appearing normal on MRI as well as regions showing complications of sickle cell disease, including focal deep white matter areas of high signal intensity (deep white matter ischemia, DWMI) seen on long TR images, focal atrophic brain areas, and infarcts. The findings in these studies are summarized as follows: Normal-appearing regions on MRI have normal MRS. In ROI including small areas of DWMI, lactate elevation was not detected, but the levels of N-acetyl-aspartate (NAA) appeared slightly elevated. In areas of DWMI 1-2 cm in size, reduced blood flow could be seen on MRA and lactate elevation could be detected with MRS. When blood flow to a DWMI region was normal, NAA was reduced and there was little lactate elevation, as cell death had already occurred. ROI consisting of atrophic tissue had reduced NAA levels but total creatine levels were not changed. Sometimes lipids, presumably from broken cell membrane, could be detected. In regions of past massive stroke, all metabolites were absent except for small amounts of lactate or lipids.


Discrepancy of xenon concentrations between end-tidal and blood collection methods in xenon-enhanced computed tomographic measurements of cerebral blood flow. Using xenon-enhanced computed tomography for the study of cerebral blood flow, simultaneous measurements of end-tidal and arterial blood xenon concentrations using the blood collection method were performed to investigate the validity of substituting the end-tidal for the arterial blood xenon concentration. Simultaneous measurement by both methods was performed 68 times in 27 patients. There was no statistical correlation between the arterial blood accumulation rate constant obtained by arterial blood and end-tidal samples, nor between the arterial blood saturation value obtained by the two methods, even when correction was made for age. In brain tissue, all parameters calculated using the end-tidal concentration were lower than those using arterial blood. We therefore suggest that cerebral blood flow values calculated using end-tidal xenon concentration are useful only for qualitative cerebral blood flow mapping, and not applicable to absolute values of cerebral blood flow.


Imaging of human T-lymphotropic virus type I-associated chronic progressive myeloneuropathies. We studied magnetic resonance imaging (MRI) of the head and cervical spine and CT of the head in 46 patients (14 men, 32 women) with chronic progressive myeloneuropathy. The findings were correlated with human T-lymphotropic virus type I (HTLV-I) serology, race, country of origin, and age. We found a female predominance of 2:1. Most patients were aged between 30 and 50 years, and most were Caribbean immigrants and black. There were 9 men and 17 women with blood antibody titers to HTLV-I and 7 men and 15 women with cerebrospinal fluid (CSF) titers. All patients with virus or antibodies in blood or CSF were Caribbean immigrants or black. T2-weighted cranial MRI showed scattered areas of high signal intensity in the cerebral white matter, usually in the periventricular and subcortical areas, but not in the posterior cranial fossa. Cranial CT revealed periventricular low density areas, ventricular enlargement, and atrophy MRI of the cervical spine showed atrophy of the cord. Myelography was normal in all 15 patients examined. No imaging differences were observed between the HTLV-I-positive and -negative patients. These findings, although consistent with demyelination, are not specific.


Cerebellar T-cell lymphoma: an unusual primary intracranial neoplasm. Primary T-cell lymphoma within the central nervous system is extremely rare. Imaging characteristics appear indistinguishable from the more common B-cell lymphoma. A case of such a primary tumor is discussed and the MRI and CT findings presented.


[Study of mediastinal lymph nodes in lung cancer using transesophageal ultrasonography]. Evaluation of mediastinal lymph nodes in patients with lung cancer is fundamental for their treatment and prognosis. Chest computed tomography (CT) is presently the most utilized diagnostic modality. In recent years endoscopic ultrasound (EUS) is being employed for this purpose. We retrospectively compared the results of CT and EUS staging of 35 selected patients with postsurgical stage. A total of 175 lymph node sites were examined. Results CT vs EUS were as follows: specificity 92% vs 98%, sensitivity 88% vs 84%, positive predicted accuracy 80% vs 96%, negative predicted accuracy 95% vs 94%, overall accuracy 92% vs 95%. The region most accessible by EUS evaluation were the paraesophageal lymph nodes; the most difficult were the right superior mediastinal nodes which cannot be imaged for anatomic reasons. EUS not only allows one to arrive at correct diagnosis with less false positive results, but also permits evaluation of lymph nodes which are not enlarged. We think that EUS, in combination with CT, is an appropriate modality for staging of mediastinal lymph nodes in patients with lung cancer.


[Surgical emergency in thyroid disease: acute respiratory failure caused by tracheal obstruction]. Diagnostic and therapeutic approach to emergency due to acute and severe respiratory compromise by thyroid goiters with tracheal obstruction is discussed. Such an alarming condition was observed in 5 (2.3%) out of 215 mediastinal goiters operated between 1967-91. Retrospective and critical analysis of these cases pointed out that preventive removal of a large goiter is the best prophylaxis; first management is not surgical, although urgent features. Admission to intensive care center and endotracheal intubation allow an adequate interpretation of the pathologic condition and operative troubles and risks. Diagnostic investigations, effective and kept to a minimum (chest x-ray, tracheo-laryngoscopy, TAC), must be carried out without delaying thyroidectomy and prolonging intubation for days. The extraction of goiter is nearly always performable by the cervicotomic way. Whenever necessary endotracheal intubation obviates the need of tracheostomy for solving transient postoperative complications such as laryngeal oedema, local hematoma and recurrent nerves stupor.


[Neoplastic adenopathies of the neck: role of tumors of the oral cavity and salivary glands]. The Authors report a series of 386 non-selected cases of cervical adenopathies with systemic (23.1%) and secondary (76.9%) tumors, treated using both simple biopsy and radical surgery. The predominant sites were right (29.0%) and left (23.6%) laterocervical. Histological tests revealed that in relation to metastatic adenopathies the predominant histotype in the series as a whole was squamous carcinoma (29.5%). Within the scope of metastatic adenopathies, tumors of the oral cavity or salivary glands, if taken together, account for the highest percentage of incidence: 14.2%. Three points emerge from an analysis of the data: 1) in the presence of lymph node tumors of the cervical district, taken as a whole, approximately 21.8% may be attributed to tumors of the oral cavity and salivary glands; 2) in the context of metastatic adenopathies, the predominant histotype is squamous carcinoma, 38.4%; 3) the presence of a high percentage of metastatic adenopathies of the neck due to cancer of the oral cavity and adjacent salivary glands always requires the greatest care to commence integrated chemo-radio-surgical treatment of prevent efficacy.


[Cholecystectomy and duodenogastric reflux]. With the aim of evaluating whether cholecystectomy causes an increase in duodenogastric reflux (DGR) 34 patient (12 males and 22 females, mean age 50 years) were examined before and 6 months after cholecystectomy. DGR was evaluated by assaying total and individual biliary acids in gastric juice and was expressed as fasting bile reflux (FBR) in mumol/h. The histology of gastric mucosa in endoscopic biopsies taken from the antrum and body was also analysed. FBR of total biliary acids rose from 2.4 mumol/h before surgery to 41.33 mumol/h after cholecystectomy (p = 0.000). A significant increase was observed for all the individual biliary acids. Histological tests of gastric mucosa revealed an increased percentage of chronic atrophic gastritis of the antrum following cholecystectomy. Histological conditions in the body were unaltered. The results of this study show that there is a significant increase in DGR (months after cholecystectomy together with increased histological damage to the mucosa of the antrum. Further studies are necessary in order to evaluate whether the two phenomena are related.


[Cancer of the rectum and sigmoid. Our experience]. The paper reports the Authors' experience during the period 1989-91 and refers to 80 operated cases of rectum-sigma cancer. Given that data reported in the literature affirm that the limit of oncological safety is 1.5-2 centimetres distal from the tumour, and owing to their use of mechanical staplers, the Authors were able to use the preferred anterior section followed by low or very low colo-rectal anastomosis in 96% of cases. Miles' technique for abdomino-perineal amputation was only resorted to in 4% of cases. Tumours parameters, short- and medium-term complications and the results of a short-term follow-up are all discussed. The Authors compare their experience with that of other researchers reported in the literature.


[Various types of small intestine occlusion. Case contribution]. The Authors examine retrospectively all the cases of obstruction of the small bowel operated at the surgical department of the Verbania Hospital in the last twenty years. The records of 341 cases, 1.4% of the total amount of the surgical operations carried out in the same space of time, were retrospectively reviewed. The Authors consider the data relating to sex, age, interval between hospitalization and operation, surgical treatment, possible previous operations, post-operative stay in hospital and complications. Having found a rate of complications significantly higher in those cases of obstruction that have required the resection of one or more intestinal loops (in all 69 cases, among them 50 cases belong to the group of the adhesions and of the strangulation), the Authors conclude by underlining the importance of recognizing and treating in time the cases where there is an irreversible vascular suffering of the bowel.


[Inguinal hernia repair with the Shouldice method. Our experience]. From 1982 to 1990, in our Surgical General Department, we have replaced the traditional technique of inguinal hernia with Shouldice's method. This choice was treatment of the following considerations: few relapses happen compared to traditional operations.


[Comparison of echo-Doppler and splenoportography in the hemodynamic evaluation of the portal system]. The Authors discuss the instrumental diagnosis of portal hypertension, comparing echo-Doppler and splenoportography by celiac artery catheterization, performed on 42 patients with cirrhosis. From the data obtained, there may be observed a clear correlation between angiographic and echographic criteria, correlated to the degree of illness. In particular echo-Doppler whether it possible, in a non-invasive way, to control the evolution of progressive haemodynamic resistance, and hepatic and splenic parenchyma, leaving to splenoportography the role to depict anatomic characteristics of portal circulation for a correct surgical program.


[Experimental evaluation, in vitro and in vivo, of the risk of infection related to the use of the most common surgical sutures]. Many parameters contribute to surgical infections. Of these, surgical sutures with their chemical and physical characteristics like tissue and cell reaction degree and bacterial adhesion, play an important role. Cell vitality, bacterial adhesion, cell reaction degree were tested "in vitro" and tissue reaction with an "in vivo" model on: plain catgut, silk, polyglycolic acid, polyglactin 910, polyglyconate, polypropylene and nylon. Vitality cell test did not reveal any cytotoxicity; bacterial adhesion showed a lower adhesion degree to synthetic materials; cell reaction showed an increase in soluble receptor (sIL 2R) values in all materials except catgut. Tissue reaction was higher for resorbable sutures, and lower for nylon. These results suggest that special attention should be paid to the choice of suture materials.


[Intraoperative colon irrigation in emergency surgical treatment of lesions of the left colon and rectum]. The Authors propose the use of intraoperative colon irrigation in emergency surgery of left-sided lesions of the colon. This technique allows the colon to be decompressed and cleaned at the same time with the possibility of performing a resection or hemicolectomy without having to resort to protective colostomy and its related problems of morbidity.


[Mechanical staplers. Personal experience]. The Authors briefly summarise their experience of using various types of mechanical staplers in abdominal and thoracic surgery with excellent results both in general and in terms of reducing operating times.


[Zenker's diverticulum in the elderly. Description of a case and surgical treatment]. The authors report a case of Zenker's giant hypopharyngeal diverticulum in an elderly patient who underwent surgery due to the severity of symptoms. This diverticulum, which is both juxtasphincteric and epiphrenal, has a pulsion pathogenesis: the presence of a hernia on the esophageal side (jato?), with which Zenker's diverticulum is frequently associated and which is often followed by reflux esophagitis, is enough to cause motor asynchronism of the crico-pharyngeal muscle which, in the presence of hypertonic conditions during deglutition, leads to the formation of a high-pressure pouch which is then responsible for the formation of the diverticulum itself. It is therefore important to check whether an associated esophageal pathology exists once Zenker's diverticulum has been diagnosed: X-ray examinations of the upper digestive tract are undoubtedly capable of identifying the presence of the diverticulum as well as other pathological associations. In the present case it was not possible to perform a sufficiently exhaustive X-ray examination in order to exclude associated esophageal pathologies. Endoscopy may be superfluous and contraindicated in cases of large diverticular pouches. Symptoms vary depending on the size of the diverticulum. A feeling of dysphagia may precede the appearance of the diverticulum, even by several years, before the onset of symptoms related to the ingestion of food: initially the patient may experience the sensation of a foreign body while eating due to the accumulation of ingested food in the diverticulum; this is followed by halitosis, sialorrhea, noisy deglutition, regurgitation of undigested food especially during sleep, and frequently bronchopulmonary symptoms "ab ingestis".(ABSTRACT TRUNCATED AT 250 WORDS)


[Gastrointestinal hemorrhage from a duodenal diverticulum]. Duodenal diverticuli are often asymptomatic and are occasionally found during the course of X-ray tests or autopsy. They are sometimes manifested in the form of symptoms related to a complication (hemorrhage, perforation, occlusion). The paper reports a case of gastrointestinal hemorrhage caused by a duodenal diverticulum which was then successfully operated. The Authors affirm that in these cases the resection of the portion of the duodenum with the diverticulum (provided the papilla is not affected) is preferable to direct hemostasis or duodenocephalopancreatectomy.


[Solitary perforated diverticulum of the right colon. Five cases]. Solitary perforated diverticulum of the right colon is a very uncommon acute disease in emergency surgery, and usually a preoperative diagnosis of acute appendicitis is performed. Five cases of solitary perforated diverticulum of the right colon are presented. Preoperative diagnostic difficulties as well as surgical procedures are discussed.


[Thoracic neurinoma: a case of a giant neurinoma]. Having observed 6 cases of benign tumours deriving from nerve sheaths, one of which was of exceptional size (19 x 11 x 10 cm), with an endothoracic localization over the past 5 years, the Authors analyse the etiopathogenetic problems, anatomo-clinical symptoms and the possible methods of treating these endothoracic tumours. Neurinomas represent between 60 and 70% of mediastinal neurogenic tumours which account for 20% of all cancers of the mediastinum. Their typical localization is the posterior mediastinum, along the paravertebral grooves, and the dimensions of these tumours vary between 3 and 8 cm, although on rare occasions they reach 15 cm. In all cases except that of the giant neurinoma, surgery took the form of straightforward enucleation. Follow-up has not revealed recidivation in any of the cases operated.


[Isolated rupture of the right hemidiaphragm with complete herniation of the liver into the thorax. Considerations of diagnosis and therapy]. The Authors report a case of right diaphragmatic hernia due to road-traffic accident. They discuss the problems of early diagnosis because it is delayed for multiple associated lesions or a low index of suspicion. The ratio of laceration of the left side to that of the right is twenty to one. The clinical and diagnostic data best able to lead to a rapid preoperative diagnosis are considered. The Authors recommended early operative treatment and the thoracic approach; thoracoabdominal incisions are more suitable whenever abdominal lesions are coexisting. The Authors stress the use of safety belts with three point fastener which have significantly decreases cranial and abdominal lesions, particularly if associated with two rolls up.


[Adrenal cystic lymphangioma. A case report]. Adrenal cysts are uncommon. A case of left adrenal cystic lymphangioma in 16 year old boy is reported. The adrenal cyst was a chance finding during radiological investigation for abdominal pain. Ultrasound and CT scan showed the cyst and suggested the correct diagnosis of cystic lymphangioma of the left adrenal gland. The patient underwent surgery and a left adrenalectomy was performed. Histology confirmed the previous diagnosis of cystic lymphangioma.


[Intrathoracic meningocele. A case report and review of the literature]. In this article the Authors describe a case of intrathoracic meningocele. That was diagnosed in a young asymptomatic woman after surgical resection. After a complete and accurate review of the literature, the Authors underline the rarity of the find while they also note the association of this pathology with Von Recklinghausen's neurofibromatosis. They also discuss etiology and clinical diagnostic aspects as well. In conclusion the Authors insist that with new diagnostic imaging techniques available today it is possible to accurately diagnose this disease preoperatively in most patients. This fact along with improved therapeutic regimens makes surgical excision almost free from the complications that were at one time observed.


Sialic acid and epithelial differentiation in colorectal polyps and cancer--a morphological, mucin and lectin histochemical study. Loss of O-acetyl substituents from sialic acid expressed in mucin secreted by hyperplastic polyps (21), adenomas (9), a mixed polyp (1) and adenocarcinomas (41) of the colorectum was investigated by mucin histochemistry (diastase PAS and mild PAS) and by lectin histochemistry (Arachis hypogaea or peanut agglutinin) with (nPNA) and without (PNA) prior neuraminidase digestion. Mild PAS and nPNA reactivity were closely correlated, indicating that loss of O-acetyl substituents at C7, C8 and C9 (hence mild PAS positive) and at C4 (hence neuraminidase labile) occur pari passu. These sialic acid alterations were characteristic of mucin secreted by both adenocarcinoma and hyperplastic polyp. The same changes occurred patchily or focally in adenoma. Five "serrated" adenocarcinomas resembled the hyperplastic polyp both morphologically and histochemically. Luminal secretions within cancers were classified as mucin-like (type I) and non-mucin-like (type II). Mild PAS was the most specific technique for mucin-like intraluminal material. However, accumulated luminal secretions (type I or II) and intracytoplasmic lumina were quite specific features of colorectal cancer and could be effectively highlighted by means of dPAS. PNA reactivity without prior neuraminidase digestion showed a distribution unlike nPNA. Whilst PNA expression was more cancer specific than either mPAS or nPNA, it was observed mainly in cancers secreting little or no mucus, thus limiting its value as a tumor marker.


Histological behaviour of chronic hepatitis in patients treated with alpha interferon. To evaluate the histological effects of alpha Interferon (IFN) therapy, serial liver biopsy specimens from 30 patients with chronic hepatitis were studied. The biopsies were examined using a scoring system. After 12 mths of IFN therapy responders were 8 out of 11 HBV infected patients, 10 out of 12 HCV infected patients and only 1 out of 7 patients with cryptogenetic hepatitis. As spontaneous improvement of hepatic changes is infrequent, our data indicate that in terms of histological patterns interferon therapy is effective in chronic viral hepatitis.


Survey of congenital tumors in perinatal necropsies. In an audit of 17,417 necropsies from 1939 to 1989 at the Royal Women's Hospital, Melbourne, Victoria, 46 congenital tumors comprising 24 teratomas, 8 vascular tumors, 6 neuroblastomas, three rhabdomyomas, two mesoblastic nephromas, one thyroid adenoma, one hepatic adenoma and one cerebellar medulloblastoma were found. The teratomas were generally large tumors and, because of their size and location, incompatible with extrauterine life. Ten teratomas were associated with polyhydramnios, three with obstructed labour, five of the fetuses were hydropic and four had malformations at sites distant to the tumor. Twenty teratomas occurred in stillbirths, half of whom were macerated. The vascular tumors were associated with polyhydramnios in three cases and hydrops fetalis in two. The neuroblastomas were all incidental findings. Four were intra-adrenal, one was an adrenal tumor which had metastasized to the liver and the remaining case was a small paravertebral lesion. Two of the three rhabdomyomas were multiple and one of these occurred in a case of tuberous sclerosis. One of the mesoblastic nephromas occurred in a hydropic fetus who had the Arnold-Chiari malformation. The thyroid and hepatic adenomas were small incidental lesions. The cerebellar medulloblastoma had led to marked hydrocephalus. Congenital tumors have different incidence, presentation and behaviour than those in childhood or adult life. The association of congenital tumors with congenital malformations and hydrops fetalis should always be remembered.


Gross handling of impalpable breast lesion specimens. The growing use of mammography has resulted in increasing numbers of impalpable breast lesion specimens requiring histopathological assessment. The examination of such specimens may be very time-consuming as multiple levels of many tissue blocks may need to be studied. Two grid localization methods are compared with a serial slicing method in an attempt to determine an efficient manner of identifying the impalpable lesion, and thus minimizing unnecessary processing. The serial slicing technique gives excellent radiological-pathological correlation, but is probably too complex for routine use. A simple cardboard and metal staple device gives very good localization, and is quick, easy, and cheap to use.


The role of the surgical pathologist in the examination of the non-palpable breast lesion. The specimen excised for the mammographically detected lesion is somewhat unique and requires special consideration by the surgical pathologist. The biopsy, in most instances, contains no grossly visible lesion and is relatively large so that blocking in its entirety is not practical. The pathologist needs to establish that the radiological abnormality is contained in the biopsy and to this end specimen radiography is required. Established prognostic parameters for breast carcinoma, such as tumor size, histological type, grade and hormone receptor status are equally applicable in the non-palpable breast lesion but consideration should also be given to the identification of epithelial proliferations which have increased risk for subsequent carcinoma. In addition, the recognition of ductal carcinoma in situ in the biopsy indicates potential for widespread disease, and lobular carcinoma in situ, a less commonly detected lesion in mammography, is associated with multifocality and disease in the contra-lateral breast. Both lesions are also associated with increased risk of recurrence in the remaining breast tissue. Failure to observe the corresponding quantity of calcium seen in radiographs relates to the fact that calcium may occur, not only as calcium phosphate, but also as calcium oxalate, the latter not being stained by hematoxylin and eosin but readily detected by its birefringent nature in polarized light. Detailed correlation of serial thin slices of the specimen with radiographic features is largely an educational exercise but is the most time-consuming step in the examination of the non-palpable breast lesion.(ABSTRACT TRUNCATED AT 250 WORDS)


Application of low magnification image analysis cytometry to prostatic tissue. Determination of the volume percentage of epithelial tissue present in prostatic tissue samples obtained by transurethral resection presents difficulties for high magnification (0.2-0.5 microns per pixel) measurement techniques due to the random orientation of tissue fragments when sectioned and the necessity of having to select "representative" areas for measurement. A low magnification (110 microns per pixel) image analysis technique was used to overcome these difficulties by allowing measurement of the epithelial tissue component to be made from the entire area of tissue available for microscopic examination. This technique which utilizes standard hematoxylin and eosin preparations was applied to 89 consecutive cases of benign prostatic hyperplasia, with measurement times of four mins per slide being obtained. Replicate measurement of 20 of these samples showed an average absolute difference between replicates of +/- 5.1 percent epithelial tissue. This method thus offers a simple, fast and practical alternative to high magnification methods of estimating the volume percentage of epithelial tissue when the selection of "representative" areas for measurement is complicated by specimen heterogeneity or when large numbers of specimens must be examined.


Computer assisted quantitation of terminal hepatic vein connective tissue in the rat. Computer assisted image analysis has been used to quantify the cellular and extracellular connective tissue component of rat liver terminal hepatic venules, in control animals and those exposed to 40% ethanol in drinking water. A significant relationship existed between the size of the terminal hepatic venule and the amount of connective tissue it contained in 14 of 15 controls and 17 of 18 ethanol exposed rats. Thickening of the terminal hepatic vein wall assessed to be present in ethanol treated rats by direct observation was confirmed by image analysis in all cases (p < 0.01). Significant differences between treated and control livers (p < 0.05) were detected by image analysis when not apparent to human observers. Sensitive quantitative assessment of terminal hepatic vein wall thickening was thus achieved by computerized analysis of liver sections.


Platelet-derived growth factor and alternative splicing: a review. The mitogenic and chemotactic potency of platelet-derived growth factor (PDGF) has linked this polypeptide to the pathogenesis of several disease states including atherosclerosis and neoplasia. We have reviewed the recent literature on aspects relating to the structure, distribution and biology of PDGF and its high-affinity cell-surface and intracellular receptors. In addition to platelets, several normal and tumor cells secrete the mitogen in one or more of three possible dimeric configurations. Alternative splicing of exon 6 in PDGF A-chain RNA results in the formation of two protein species with different carboxy-termini. Initially, it was thought that the longer A-chain variant was processed only by transformed cells. However, recent evidence indicates that alternative splicing occurs in several cells which express the A-chain, including early Xenopus embryos. The functional significance of the exon 6 product, a highly basic region spanned by 18 amino acid residues (A194-211), is not precisely clear. We have summarized recent findings which implicate roles for A194-211 in the processing, secretion, and mitogenesis of the A-chain homodimer, nuclear transport signalling, and heparin binding. Thus, alternative splicing could play an important role in the modulation of the functional properties of the PDGF A-chain variants per se and in the complex interactive network of polypeptide growth factors and cytokines.


A method for enriching myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from human cord blood by accessory cell depletion. Human cord blood provides a convenient alternative to bone marrow as a rich source of hemopoietic progenitor cells. This study reports a simple means for enriching a cord blood progenitor cell population by accessory cell depletion. Two methods of monocyte depletion were tested. A Cytodex 3 microcarrier system using collagen coated dextran beads was compared to the more commonly used method of plastic plate adhesion. The method of plastic plate adhesion gave a significantly higher cell recovery. T cell depletion using a recently characterized rat monoclonal antibody which fixes human complement was also investigated. A combined method of monocyte depletion by plate adhesion and T cell depletion resulted in the removal of > 96% of monocytes and > 98% of T cells. This led to a significant enrichment of myeloid (CFU-GM) and erythroid (BFU-E) colony growth. Such enriched progenitor cell populations provide a useful starting population for any study on hemopoiesis.


Effects of cholera toxin on human colon carcinoma cell lines. This study reports on changes in morphology and membrane transport in 5 human colon carcinoma cell lines treated with cholera toxin (CT). Three of the cell lines that grew as monolayers (LIM 1215, LIM 1899, LIM 2099) and 1 that grew as floating clumps (LIM 2408) did not show morphological changes after CT treatment. However, cell line LIM 1863 that grows as floating "crypt-like" organoids showed rapid and distinctive changes in morphology and membrane transport after CT treatment. At 1 and 6 hrs after CT treatment, light and transmission electron microscopy revealed rapid dilatation of the central lumen of organoids and the appearance of 2 populations of apical vesicular inclusions. The first population was unusual in being non-membrane bound and limited by fuzzy filamentous material. The second population was membrane bound. Scanning electron microscopy at 1-6 hr after CT treatment showed swelling and loss of surface microvilli on some, but not all, cells. At 24 hr after CT treatment the majority of organoids showed evidence of fluid accumulation and small apical vesicles coalesced to form large single vacuoles that obliterated normal cell morphology. By 48 hr, continued swelling produced extreme attenuation of the plasma membrane with cells taking on an "endothelial cell-like" appearance. The response to CT was dose-dependent. Uptake studies using 86Rubidium and blocking studies using ouabain and amiloride indicated that CT is acting on the Na+/K+ ATPase membrane pump to cause the increased fluid uptake by LIM 1863 cells. This study is the first to report specific morphological changes in intestine-derived cells in response to CT.(ABSTRACT TRUNCATED AT 250 WORDS)


The putative melanoma tumor-suppressor gene on human chromosome 6q. The tumorigenicity of malignant melanoma cells may be suppressed experimentally by the introduction into these cells of human chromosome 6 or mouse chromosome 4. These chromosomes share a homologous region, contained in human chromosome 6q12-21. Abnormalities of this human chromosomal region have been found frequently not only in cutaneous and uveal malignant melanomas, but also in a range of other tumors. In all these, mutations of tumor-suppressor genes on human chromosome 6q may be involved. Identification of this putative tumor-suppressor gene may give new insights into the biology of malignant melanomas, and could pave the way for new treatment for such tumors, based upon the tumor-suppressor protein which this gene is likely to encode.


Myositis ossificans in childhood.

Myositis ossificans in childhood. Myositis ossificans is a benign, localized, ossifying lesion of soft tissues that is rarely reported in young children. This paper describes two cases found in a search of the surgical biopsy files of the Adelaide Children's Hospital over the 30 yr period from 1962 to 1991, in boys both aged 7 yrs. Diagnosis was assisted by combined clinical, radiological and histopathological information (including an adequate well-orientated biopsy in Case 1 that demonstrated the characteristic growth pattern) enabling differentiation from other possibilities such as fibrodysplasia ossificans progressiva.


DNA cleaving modes in minor groove of DNA helix by esperamicin and calicheamicin antitumor antibiotics. This study examines and compares DNA cleavage modes by several esperamicin derivatives and calicheamicin. We found that the deoxyfucose-anthranilate moiety is a key factor to determine their DNA cutting modes. Probably, the bulky moiety hinders the abstraction of hydrogen atom from deoxyribose by the C-1 carbon radical of phenylene diradical. On the basis of the experimental results, detailed DNA cleaving modes in DNA minor groove by esperamicin and calicheamicin have been discussed.


Molecular mechanisms for controlling spontaneous and induced mutagenesis. Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low. We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis. We also deal with suppression of alkylation-induced mutations by O6-methylguanine-DNA methyltransferase.


Facile and stereoselective synthesis of 2'-5'-oligothioadenylate by UO2(2+) ion catalyst. The synthesis and characterization of 2'-5'-oligothioadenylate by UO2(2+) ion catalyst are described. The polymerization of imidazole-activated thioadenylate or thioinosylate yielded oligomers containing mainly 2'-5'-internucleotidyl blond and Rp configuration at phosphorous atom.


Facile reductive cleavage of purine nucleosides to acyclonucleosides using diisobutylaluminium hydride (DIBAL): a new synthetic method for the preparation of 9-ribitylpurine derivatives. Reaction of purine nucleosides, such as 2',3'-isopropylideneinosine (1a) and 2',3'-isopropylideneadenosine (1c), with diisobutylaluminum hydride (DIBAL) in dry tetrahydrofurane resulted in the formation of the corresponding 9-(2',3'-isopropylideneribity)purines (2) in good yields. Oxidation of the ribityl derivatives (2) with NalO4 and subsequent reduction with NaBH4 gave the corresponding 9-(2',3',4'-trihydroxybutyl)-purine derivatives (4) in high yields. Deprotection of compounds 2 and 4 in 80% acetic acid gave the corresponding 9-(2',3',4'-trihydroxybutyl)purines (5) and 9-ribitylpurines (6), respectively.


Regioselective halogenation of some 2,3-anhydro pyrimidine nucleosides with dilithium tetrahalocuprates. The reaction of 1-(2,3-anhydro-5-O-trityl-beta-D-lyxofuranosyl)-2-O-methyluracil (1a) and its thymine analogue (1b) with dilithium tetrahalocuprate (Li2CuX4) revealed excellent to perfect regioselectivity, yielding 2,2'-anhydro-3'-halonucleosides (2a-d), while the same reactions with 2,3-anhydro uracil and thymine nucleosides (4a,b) gave arabinosyl (5a-d) and xylosyl halohydrins (6a-d) with the respective product ratio of 7:3 to 8:2. compounds 5 and 6 were isolated as the 2-O-(7) and 3- O-mesyl derivatives (8).


Synthesis of 2'-deoxy and 2',3'-dideoxynucleoside derivatives from thioglycosides. The synthesis of both 2'-deoxy and 2',3'-dideoxynucleoside derivatives by the reaction of thioglycosides with nucleoside bases was examined. The stereochemical outcome at the anomeric position was found to depend on the protecting groups and the C-3 configuration in the sugar moiety, the kind of activator, and the reaction temperature. Based on these findings, 2'-deoxy-D-xylo nucleoside and 2',3'-dideoxynucleoside derivatives have been synthesized in beta-selective manner.


The synthesis of cyclopropano nucleosides. A tricyclic, fused cyclopropano nucleoside 8 containing a ketal group was synthesized by the one-pot seven sequential reactions of a trimesylated allofuranosyl adenine derivative 6 with Mg(OMe)2. When KOH was used instead of Mg(OMe)2, an alpha, beta-unsaturated ketonucleoside 7 was obtained.


Synthesis and interaction studies of watersoluble nucleic acid analogs containing serine as a spacer. Watersoluble nucleic acid analogs containing L- or D-serine as a spacer were synthesized. Thymine was used as nucleic acid bases of these analogs. The base contents of these analogs were 93-94%. These analogs were found to form stable polymer complexes with Poly A or DNA by specific base-base interaction, which were observed from hypochromicity of UV spectra. In both cases, the maximum hypochromicity values of PEI-L-Ser-Thy were higher than that of PEI-D-Ser-Thy.


Synthesis and properties of oligodeoxyribonucleotides containing a mutagenic base, N4-aminocytosine. Oligodeoxyribonucleotides containing a mutagenic base analog, N4-aminocytosine, 5'-AATTGC(am)AATT-3' and 5'-AATTAC(am)AATT-3' (C(am); N4-aminocytosine) were prepared by chemical modification of 5'-AATTGCAATT-3' and 5'-AATTACAATT-3', respectively. The values of Tm were 29 degrees C for 5'-AATTGC(am)AATT-3' and 32 degrees C for 5'-AATTGCAATT-3'. In contrast, no melting was observed for 5'-AATTAC(am)AATT-3' and 5'-AATTACAATT-3'. These data show that the stability of C(am)-purine paris is C(am)-G > C(am)-A and that C(am)-G is less stable than C-G. This property is consistent with the incorporation specificity of N4-amino-dCTP during DNA synthesis in vitro.


Synthesis and property of alpha-oligodeoxynucleotide phosphorothioate. Unnatural DNA analog, alpha-2'-deoxy-oligoadenylate phosphorothioate bearing 3'-normal monophosphate moiety was conveniently synthesized through phosphoramidite solid support method. The starting material of the DNA analog, alpha-2'-deoxyadenosine, was prepared in a stereospecific manner by the coupling reaction between activated nucleobase and 1-chloro-2-deoxyribose derivative.


The influence of oligodeoxyribonucleotide phosphorothioate pyrimidine strands on triplex stability. The analogues of the homopyrimidine oligonucleotide dT15 has been synthesized. The analogues, contains phosphorothioate bonds of a mixture of diastereoisomers or one of the two stereoisomer (either Rp or Sp). The analogues were mixed under conditions conductive to the formation of triple stranded assemblies. The mixtures were characterized by their thermal stabilities (Tm values) and CD spectra.


Interaction of antisense DNA with nucleic acids/proteins. In order to study interaction of various types of labeled antisense DNAs were prepared. Fluorescein and 2,2,6,6-tetramethypiperidine-N-oxyl were the label molecules, which were introduced to 5'-end of oligonucleotides and their analogs. Interactions of labeled antisense DNAs with nucleic acids or proteins such as HSA, HIG and TF, were studied by UV, fluorescence depolarization spectroscopy, and ESR spectroscopy. Hybrid formation of antisense DNAs with oligonucleotides in solution could be monitored by the increase in fluorescence anisotropy (r) and by intensity change in ESR spectra. When phosphorothioate type antisense molecules anchoring fluorescein (F-OPT) were mixed with proteins, r drastically increased, whereas ODN slightly increased. These results suggest that OPTs have much more affinity for proteins than ODNs.


Syntheses and properties of fluorescent labeled oligonucleotides containing deoxyethenoadenosine at 5' end. The fluorescent labeled oligodeoxyribonucleotides which contain deoxyethenoadenosione (d epsilon A) at their 5' end were prepared by treating CPG bound oligonucleotides with 5'-DMTr-deoxyethenoadenosine-3'-H-phosphonate. The hybrid formation of d epsilon A-oligonucleotide with its complementary DNA was studied by fluorescence spectroscopy. The fluorescence of d epsilon A in a single strand was largely quenched by stacking interaction with the base at 3' position. When d epsilon A-oligonucleotides hybridized with their complementary strands, relative fluorescence quantum yields (Qrel) against d epsilon A changed in specific manners. These results suggest that d epsilon A-oligonucleotides are applicable to study the local structure of DNA in solution.


Kinetics and thermodynamics of double-helix formation of self-complementary deoxyribooctanucleotides d(TCTATAGA) and d(TAGATCTA). Double-helix formation of self-complementary deoxyribooctanucleotides, d(TCTATAGA) and d(TAGATCTA), with identical nearest neighbor base pairs has been studied by means of UV melting and temperature-jump techniques. The self-complementary duplexes of both octanucleotides with identical nearest neighbors had similar stabilities: The stabilization energies of the octanucleotides at 25 degrees C were 5.8 kcal mol-1 for d(TCTATAGA) and 6.7 kcal mol-1 for d(TAGATCTA). On the kinetic curve the melting reactions finished within 20 ms for d(TCTATAGA) and 40 ms for d(TAGATCTA) at 20 degrees C. For both octanucleotides the rate constants of dissociation increased and the rate constants of association decreased with increasing temperature.


Novel strategies towards construction of non-ionic and achiral backbones in antisense oligonucleotides. A convenient synthesis of a thymidine (T) nucleoside dimer (T-3'-CH2-O-NCH3-CH2-4'-T) 12 has been accomplished via a nucleoside coupling reaction. An alternative synthesis of 3'-deoxy-3'-C-hydroxymethyl-thymidine is described. The new dimer and methodology is useful for the development of backbone-modified antisense oligonucleosides.


Fluorescent-labeled oligonucleotides that exhibit a measurable signal in the presence of complementary DNA. Oligonucleotide derivatives with a fluorescent dye were designed for exhibiting a measurable signal only when they bind to complementary DNA in aqueous solution. The oligonucleotide with a dansyl group at the specific 2'-sugar residue was synthesized by using the protected 2'-dansylaminouridine phosphorobisamidite. The dansyl-oligonucleotide conjugate binds to its complementary DNA to form duplex with a normal stability and exhibits enhanced fluorescence together with a blue-shift in emission maxima after the hybridization. Another possible candidate involved the use of pyrene-excimer emission upon forming ternary complex between two pyrene-labeled oligonucleotide probes with target DNA. A new and general method for introduction of a pyrene fluorophore into the 3'- or 5'-terminal hydroxyl group of oligonucleotides via different linkers was developed.


Separation and characterization of diastereoisomeric oligonucleotide. For developing the antisense method, two types of oligonucleotides suitable for antisense molecules were explored for their essential properties. One is oligonucleoside phosphorothioate, and all their possible R/S diastereoisomers were separated and purified by reversed phase liquid chromatography. Isolated diastereoisomers have been investigated for the correlation between their configuration and hybridization manner. Separation, R/S characterization and spectroscopical properties of these oligonucleotides will be discussed.


Double-helix formation of self-complementary chimeric oligonucleotides r(CG)nd(CG)3-n (n = 0, 1, 2, 3). Double-helix formations of self-complementary chimeric hexanucleotides, r(CGCGCG), r(CGCG)d(CG), r(CG)d(CGCG), and d(CGCGCG), have been studied spectrophotometrically an thermodynamically in 1 mol dm-3 NaCl buffer. CD (circular dichroism) spectra showed that r(CGCGCG), r(CGCG)d(CG), and r(CG)d(CGCG) formed A-type double helix, while d(CGCGCG) formed B-type double helix. The stabilization energies of these helices at 37 degrees C obtained from UV melting analyses were 9.2 kcal mol-1 for r(CGCGCG), 8.2 kcal mol-1 for r(CGCG)d(CG), 6.8 kcal mol-1 for r(CG)d(CGCG), and 8.5 kcal mol-1 for d(CGCGCG), respectively.


Purification and characterization of tRNA(adenosine-1-)-methyltransferase from Thermus thermophilus HB27. A58, the conserved adenosine residue in the T psi C loop of tRNAs, is methylated to m1A 58 in an extreme thermophile, Thermus thermophilus HB27. The enzyme catalyzing this methyltransfer reaction was purified from the thermophle. The substrate specificity of the enzyme was investigated by using tRNA fragments. The enzyme can transfer the methyl group to the 3'-half fragment of E. coli initiator tRNA, indicating that the main recognition site of the enzyme exists in the 3' half of tRNA including the T-loop and the T-stem.


Cloning and sequence analysis of the evgAS genes involved in signal transduction of Escherichia coli K-12. We have cloned and sequenced new Escherichia coli genes which belong to member of the family of environmentally responsive two-component system and named evgA and evgS because their amino acid sequences were found the most homologus to the Bordetella pertussis bvgA and bvgS. They were mapped at 51 min. and extending from 6B9 to 7G9 in the Kohara miniset library of the E. coli chromosome. In fact, both EvgA and EvgS proteins predicted from their DNA sequences were identified in the in vitro coupled transcription translation system. When the evgA and evgS were expressed on multiple copy plasmid in an envZ deletion strain, ompC expression was also regulated by temperature, MgSO4 and nicotinic acid, by which virulence of Bordetella pertussis is controlled via BvgA and BvgS. These results indicate that ompC expression was controlled by in vivo cross-talk via EvgA and EvgS which can work in E. coli the same way as BvgA and BvgS.


Activities of HIV-RNA targeted ribozymes transcribed from a 'shot-gun' type ribozyme-trimming plasmid. We have constructed a "shot-gun" type ribozyme-trimming system. By concatenating several units, each consisting of a trans-acting ribozyme (targeted to HIV-RNA) and cis-acting ribozymes (trimming 5'- and 3'-ends of the trans-acting ribozyme), several kinds of trans-acting ribozymes can be liberated upon transcription and self-cleavage. Since each liberated HIV-RNA-targeted ribozymes can work independently, they can simultaneously cleave HIV-RNA at several different sites. Ribozymes were targeted at relatively conserved GUC-containing sites at LTR, gag and tat regions.


DNA conformation of the region preceding the beta-lactamase promoter of pUC19 which is required for efficient transcription. The upstream region of the beta-lactamase promoter of Escherichia coli plasmid pUC19 has a DNA curvature (bent DNA). This region was replaced with another sequence by using randomly synthesized oligonucleotides. Among the reconstructed plasmids, a plasmid which could endow E. coli cells with the strongest resistance to ampicillin on plates was selected. Nucleotide sequence and DNA conformation of the altered region was investigated.


Quantification of the c-myc gene in gastric carcinomas by the triplex polymerase chain reaction and high performance liquid chromatography. The triplex polymerase chain reaction (PCR) and high performance liquid chromatography (HPLC) techniques were used to examine the state of amplification of the c-myc gene in gastric carcinomas. Sequences from the c-myc gene and from the two control genes were coamplified by PCR. The coamplified PCR products were separated and quantified by HPLC and the copy numbers of the c-myc gene were calculated by comparing the peak areas generated by PCR products. Increased copy numbers of the c-myc gene were found in 2 of 5 patients.


Efficient cDNA cloning method using solid phase DNA probe. We are now developing a novel and efficient method using solid phase DNA probe to isolate a particular recombinant cDNA from single stranded cDNA library. Target clone coding metapyrocatechase (MPC) and cDNA library constructed from mRNA of U-937 (human lymphoma cell line) were converted to single stranded form by superinfection of helper phage (M13KO7). Probe DNA (25 mer) composed of a portion of the target cDNA was synthesized, attached to an HPLC gel and used as a solid phase DNA probe. Hybridization between probe DNA and target clone was performed in an Eppendorf tube within a few hours. Competent cell (JM109) was transformed with about one-twentieth of hybridized and eluted fraction by Hanahan's method. From the mixture of 1 ng of MPC vector and 5 micrograms of cDNA library, we obtained 50 colonies containing MPC gene out of 63 transformed colonies.


Topological alteration of plasmid DNA during cell growth of Escherichia coli. Superhelical alteration of plasmid DNA in Escherichia coli cells was observed during cell growth. Plasmid DNAs were being supercoiled while the cells were actively dividing, and they were partially relaxed after the cultures reached stationary. Unknown structural materials of plasmid DNA which migrated faster than usual superhelices in agarose gel electrophoresis were found among the strains regardless of gyrase genotype.


Relationship between the 5'-leader sequence of mRNAs and the translation efficiency in a eukaryotic in vitro system. To examine the effect of the 5'-leader region of mRNAs in regulating translation efficiency, chimeric mRNAs which have various sequences of the 5'-leader were synthesized by the SP6 in vitro transcription system, and translated in the wheat germ in vitro translation system. Translation efficiency was affected by the length of the 5'-leader and the number of the codon A-U-G at the translation initiation site.


Tachyplesin I as a model peptide for antiparallel beta-sheet DNA binding motif. In this study, we present a model compound for antiparallel beta-sheet-DNA interaction. Tachyplesin I, cationic antimicrobial peptide, interacts through contacts with the minor groove. Secondary structure of tachyplesin I, antiparallel beta-sheet constrained by two disulfide bridges and connected by beta-turn, contributes significantly to its DNA binding. The present results give valuable information for design of sequence-specific DNA binding peptide based on antiparallel beta-sheet.


Interaction of a protooncogene product, Myb with DNAs. The DNA-binding domain of Myb consists of three imperfect tandem repeats and the third one which is essential for sequence-specific binding was established to have a helix-turn-helix-related motif. DNA sequences recognized by Myb have been reported to contain TAACPy sequence. Here we have examined the details of Myb-binding sequence. Using DNAs with a single mutation on the various sites of two specific DNAs and some fragments of the DNA-binding domain of Myb, we have found that (i) in a specific DNA which contains only one AAC sequence, each AAC nucleotide is found to be essential for the specific binding of Myb, while any other mutations cause no serious binding loss, (ii) in a specific DNA which contains two AAC sequences separately, one AAC is not so important in the binding, and (iii) for the specific binding with DNA, at least both repeats 2 and 3 of Myb are required. These findings suggest that repeat 3 containing a helix-turn-helix-related structure recognizes the core AAC sequence and repeat 2 supports this recognition by interactions with phosphate groups of DNA.


Effects of base damages on DNA replication--mechanism of preferential purine nucleotide insertion opposite abasic site in template DNA. DNA polymerase preferentially inserts purine nucleotides opposite non-instructive lesions such as abasic sites during DNA replication. In order to elucidate the mechanism of the preferential insertion, a DNA template containing a model abasic site and primers containing 4 different nucleotides (A,G,C,T) at primer terminus were synthesized. The stability of the primer terminus nucleotide placed opposite the abasic site was evaluated on the basis of its sensitivity to 3'-5' exonuclease associated with DNA polymerase.


A study on conformationally restricted sangivamycins and their inhibitory abilities of protein kinases. Conformational restrictions of sangivamycin, a rather selective inhibitor of PKC, could be achieved by the use of the steric effect and the gauche effect of the substituents on the ribofuranose moiety. The conformational deviations obtained by these methods were found to nicely correlate with the inhibitory ability of PKC.


On the base sequence of the promoter of Alzheimer's disease amyloid A4 precursor gene in transcription initiation. The base sequence of a specific DNA region of the APP gene identified as the promoter was analyzed by means of the quantity Sr corresponding to superdelocalizability of oxygen ion of each phosphate for the ten DNA dimer units (XY/Y' X') and the six [(XY/Y'X')+H+] complexes. The base sequence of the -35 signal was determined to be 5'-CAGCTG-3' (-49 to -44). On the other hand, the base sequence of the -10 signal was found several ones. Therefore, transcription is to initiate at multiple sites. The mechanisum how RNA polymerases can recognize each transcription site, was proposed. The result explains fairly well some characteristics of the promoter of the APP gene experimentally found so far.


Recognition and structural perturbation of GC box DNA by Sp1 zinc finger. Interaction of Sp1 with GC box DNA was investigated by several footprinting experiments. Methylation of four guanine bases is strongly protected by Sp1 binding, while one guanine base in GC box is extremely hypermethylated. Sp1 binding also induces new cleavage at 5'-GA-3' site within GC box by bleomycin-iron complex.


Mutations induced by DNA lesions in hot spots of the c-Ha-ras gene. In order to investigate whether several DNA lesions (O6-methylguanine, 8-hydroxyguanine, xanthine, an abasic site analogue and hypoxanthine) activate a c-Ha-ras gene and to determine the type of mutations induced by the DNA lesions, they were introduced into a synthetic c-Ha-ras gene by DNA cassette mutagenesis techniques. The modified genes were transfected into mouse NIH3T3 cells and the c-Ha-ras genes present in transformed cells were analysed. O6-methylguanine and xanthine induced a mutation to A, hypoxanthine induced a mutation to G. 8-hydroxyguanine and the abasic site analogue caused random mutations in the modified and adjacent positions. These results indicated that the synthetic c-Ha-ras gene is very useful for the detection of mutations caused by a DNA lesion.


Photosensitized formation of 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in DNA by riboflavin. Potosensitized formation of 8-hydroxyguanine in DNA by riboflavin was observed. A reaction mechanism involving guanine radical cation and hydration reaction was proposed. This hypothesis was confirmed by the incorporation of [18O]-atom within guanine moiety in isotopic experiments using [18O]-H2O. Photosensitized formation of oh8Gua by riboflavin was also observed in cellular DNA.


Post-translational modification of protein by tyrosine sulfation: active sulfate PAPS is the essential substrate for this modification. In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.


Some novel pyrimidine nucleoside rearrangements effected by diethylaminosulfur trifluoride (DAST). Pyrimidine nucleosides (or their 5'-aldehydes) when treated with DAST give O2,5'-(fluoro)-anhydronucleosides. If this is prevented by blocking N-3 or O4, the desired 5'-deoxy-5'-(di)-fluoronucleoside is accompanied by the production of a compound resulting from migration of the base following scission of the N-1-->C-1' bond and formation of O2-->C-5'. This is a particular example of a much more general phenomenon, seen when suitably substituted ribofuranoses are treated with DAST.


Mechanism of protein priming DNA replication of B.subtilis phage M2. B.subtilis phage M2 uses a protein, instead of RNA, as the primer of its DNA replication. Hence this protein encoded in the phage genome is called as the primer protein (PP). At the initiation of DNA replication, a hetero dimer complex with its own DNA polymerase and the PP supposed to interact with the terminal protein (TP), which is covalently bound to the template DNA (TP-DNA). PP contained an important adhesive amino acid sequence, Arg-Gly-Asp (RGD), near the carboxyl terminal. We have recently showed that the synthetic RGD peptide inhibited the transfection of phage M2. By site-directed mutagenesis, we introduced different amino acid into the RGD site of PP. These altered PP decreased obviously the priming activity in vitro.


Action of 5-trifluoromethyl-2'-deoxyuridine on DNA synthesis. The action of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) on DNA synthesis was investigated in vitro assay systems with purified DNA polymerases. CF3dUrd was incorporated into the DNA of mammalian cells in culture. We studied the incorporation of CF3dUrd 5'-triphosphate (CF3dUTP) into DNA and effect of CF3dUrd residue on DNA synthesis. Therefore, we synthesized oligonucleotides that allow site specific introduction of a CF3dUrd residue into a synthetic DNA oligonucleotide. After CF3dUTP incorporation, the primer was extended for human DNA polymerase alpha (pol. alpha). When CF3dUrd residue was located at an internucleotide site in the template, however, pol. alpha was exhibited a strong arrest band one nucleotide after the CF3dUrd residue site, and Escherichia coli polymerase I (Klenow fragment) also exhibited a weaker arrest band one nucleotide before the CF3dUrd residue. These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.


Synthesis of chimeric RNAs between U6 small nuclear RNA and (-)sTRSV and analysis of their cleavage activities against the substrate RNA. U6 small nuclear RNA (U6 snRNA) is one of the spliceosomal RNAs essential for pre-mRNA splicing. Highly conserved region of U6 snRNA shows a structural similarity with the catalytic center of the negative strand of the satellite RNA of tobacco ring spot virus [(-)sTRSV], supporting the hypothesis that U6 snRNA has a catalytic role in pre-mRNA splicing. To test this hypothesis, we examined in vitro whether synthetic RNAs consisting of the sequence of the highly conserved region of U6 snRNA or various chimeric RNAs between the U6 region and the catalytic center of (-)sTRSV could cleave a substrate RNA that can partially base-pair with them and has a GU sequence between the pairing regions. Chimeric RNAs with 70 to 83% sequence identity with the conserved region of S. pombe U6 snRNA cleaved the substrate RNA at the 5' side of the GU sequence. In addition, we found that the highly conserved region of U6 snRNA is similar in structure to the catalytic core region of the group I self-splicing intron in cyanobacteria. These results support the hypothesis that U6 snRNA catalyzes the pre-mRNA splicing reaction and U6 snRNA may originate from the catalytic domain of an ancient self-splicing intron.


Sequence specific block of in vitro DNA synthesis with isopropyl phosphotriesters in template oligodeoxyribonucleotides. We have synthesized four oligodeoxyribonucleotides each bearing an isopropyl phosphotriester at a defined position. These oligomers were used as templates for in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I large fragment. Results showed that the phosphotriester inhibits the DNA chain elongation and the level of the inhibition is dependent on the base 5' to the phosphotriester.


Temperature-gradient DNA probe chromatography of nucleic acids on nonporous supports. Temperature-gradient DNA probe chromatography of nucleic acids on a nonporous support with a homogeneous particle size of 2.5 microns showed a higher base sequence discriminating power and a larger linear capacity than that on a porous support with a larger and less homogeneous particle size. The resolution on the nonporous support was high enough to separate samples with a single-base mismatch of the less destabilizing groups, including G-G and G-T base mismatches, even when it locates very near the end of the probe.


The formation and enzymatic repair of DNA modifications caused by the haloethylnitrosoureas and related compounds. The haloethylnitrosoureas are both useful antitumor agents and known carcinogens. These biological activities are believed to be associated with DNA modification, and some biologically significant lesions have been identified in DNA exposed to these agents. At the same time, DNA repair is a cause of resistance to treatment by these agents, and may also serve as protection against their carcinogenic effects.


DNA hydrolysis by rare-earth metal ions. Plasmid DNA and poly(dA) are cleaved by rare-earth(III) ions at pH 7-8 and 50 degrees C. The cleavage has been confirmed by prompt conversion of supercoiled pBR 322 plasmid DNA (Form I) to a relaxed Form II. Furthermore, degradation of poly(dA) to shorter oligonucleotides is clearly evidenced by HPLC. A possible application of the metal ions (and their complexes) to artificial nucleases is indicated.


DNA strand scissions by hydroxamic acids in the presence of Cu(II) ion under aerobic conditions. Carbazolyloxyacetohydroxamic acid (1), 9,9'-decamethylene-bis-carbazolyloxyacetohydroxamic acid (2), benzohydroxamic acid (3), and acetohydroxamic acid (4) without reducing agent under aerobic conditions induced DNA strand scissions with increasing activities in the order of 4 > 3 > 2 > 1. The inhibition experiments indicated that hydrogen peroxide and superoxide participated in the reactions, but hydroxyl radical or singlet oxygen did not.


Identification of important bases for the self-cleavage activity at two single-stranded regions of genomic HDV ribozyme. In order to determine important bases at two single-stranded regions [SSrA (726-731 nt) and SSrB (762-766)] derived mainly from secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several point mutants at these two regions on the HDV88 molecule (683-770). Among the bases at SSrA and SSrB regions C763 was found to play an essential role during self-cleavage process since substitutions to any other bases viz. A or G or U completely abolished the activity.


Ribozymes for specific inhibition of mRNA function in the nematode Caenorhabditis elegans. Nine different hammerhead ribozymes were designed for three specific sites of unc-22 mRNA in C. elegans, which carry the common catalytic core and 12, 16 or 20 flanking nucleotides for base pairing with the mRNA, and tested for cleavage of short substrate RNA in vitro. All the ribozymes cleaved the substrate RNA catalytically at 37 degrees C and the activities at 37 degrees C were higher for all the ribozymes than those at 20 degrees C, the nematode growth temperature. Plasmids carrying each of a few different promoters and lacZ reporter gene were prepared and tested in the nematode as a test of vectors for the expression of ribozymes in vivo.


Cleavage of DNA by viologen related compounds and their incorporation into oligodeoxyribonucleotides. The DNA cleavage reaction by viologen and related compound such as 2,7-diazapyrenium salt was investigated. These viologen analogues were successfully incorporated into the oligothymidylate in the form of covalent bonding at the site of the phosphorous backbone through the linker arm.


Recognition of (d(TTTATT) and d(TTATTT) by capillary affinity gel electrophoresis (CAGE) using poly(9-vinyladenine)-polyacrylamide conjugated gel. Sequence-specific recognition of oligodeoxynucleotide isomers (TTTATT and TTATTT) was achieved by using polyacrylamide-poly(9-vinyladenine) conjugated gel filled capillary affinity gel electrophoresis. It was found that the interaction between poly(9-vinyladenine) and the isomers was dependent on the sequential thymidylic acids of them.


NMR studies on solution structure of single-stranded oligonucleotides causing line broadening. Unusual line broadening of 1H-NMR lines attributable to the proton (8H) of guanine residues was observed for all tetradeoxyoligonucleotides tested here which have a specific base sequence of dGXXG (X = A or T). For the same samples, line broadening was also obtained in the 31P-NMR spectra. These broadened signals did not become sharp up to 60 degrees C. This unusual spectral phenomenon has been attributed by 2D-NMR and differential NOE to the compact solution structure of the oligonucleotides.


Pentacoordinate oxyphosphorane intermediate always exists in aqueous solution. Gas-phase ab initio calculations indicate that dianionic pentacoordinate oxyphosphoranes do not have a kinetically meaningful intermediate. The simplest oxyphosphorane PO5H3(2-) has the least tendency to have a pentacoordinate intermediate. However, it does have a pentacoordinate intermediate when it is solvated with six water molecules. These results support the hypothesis that the phosphoryl transfer reactions take place via pentacoordinate intermediate not only in acidic but also in basic media.


Assignments of Raman tensors to Raman bands of nucleic acids: use of a single crystal of adenosine triphosphoric acid and DNA fibers. A polarized laser (488.0 nm) Raman spectroscopic measurement has been made of a single crystal of adenosine triphosphoric acid trihydrate (ATP.3H2O), whose space group is P2(1)2(1)2(1), and whose dimensions are about 10 microns x 100 microns x 100 microns along the crystallographic a, b and c axes, respectively. For each Raman band in the 300-1800 cm-1 range, relative scattering intensities, Ibb and Icc, which correspond to the bb and cc components of the Raman tensor of the crystal, have been determined. In an aqueous solution of ATP, the depolarization ratio of each Raman band has also been determined. On the basis of these experimental results, some discussions are given of the shapes and orientations of the Raman scattering tensors localized in the base residue, ribose portion, and phosphodiester portions. A similar type of study has been made with a few DNA fibers.


Dynamical conformational change of DNA induced by synthetic polymers: direct observation by fluorescence microscopy. Brownian motion and Higher-order structure of DNA molecules complexed with synthetic polymers: poly-L-arginine (pArg) and polyacrylamide (pAAm), were observed using fluorescence microscopy. Quantitative analyses of the conformation and motion of the complexes demonstrate that these two polymers influence the structure of DNA molecules in an opposite way. The electrostatic binding of pArg induces contraction of DNA, whereas the intercalative pseudo-grafting of pAAm extends DNA.


Photochemical deoxyribose C2' oxidation in 5-iodouracil-containing hexanucleotide. To investigate photochemistry of 5-iodouracil (IU) in DNA, photoreaction of IU-containing oligonucleotides was examined. It was found that d(GCAIU G C) 2 undergoes selective photochemical C1' and C2' oxidation at the 5' side of IU residue to provide ribonolactone-containing hexamer 1 and erythrose-containing hexamer 2. Upon heating under alkaline conditions, erythrose-containing hexamer 2 was found to undergo retro aldol condensation to provide two fragments having glycolaldehyde termini.


Interaction of novel tris-intercalators with DNA. Spectrofluorometric studies. Novel DNA binding ligands (1 and its stereoisomer 2) which contain three potentially intercalating units in a linear molecular skeleton were prepared. From a study of the displacement of ethidium bromide from several natural and synthetic polynucleotides, both compounds 1 and 2 were found to show an AT base-pair preference in the interaction with DNAs with 2 a slightly higher affinity for DNA. This result is in sharp contrast to that for acridine and anthraquinone, because these two compounds exhibit a GC-preference.


Electronic interaction between 3'-azido-2'-deoxythymidine and nucleic acid bases by fluorescence spectroscopy. The ultraviolet (UV) absorption and fluorescence nature of the mixtures of 3'-azido-3'-deoxythymidine (AZT), poly 1, N6-ethenoadenylic acid (poly, epsilon A) and mixtures of AZT and poly A (AZT+poly epsilon A) at various molar ratio has been studied. On the basis of the present results, it may be concluded that the azide group of AZT (N6', N7', and/or N8') may link to the phosphate groups of polynucleotide. Thus, results obtained suggest that there are electronic interaction between thymine and ethenoadenine lings at the first excited singlet state.


Preparation of thianucleoside derivatives. 1'-Deoxy-4'-thioribonucleoside 1 was synthesized from acyclic allylic alcohol 2 by 8 steps. TMSOTf and SnCl4 were found to be a good mediator for the key coupling of thiafuranose part with pyrimidine base to give beta-isomer preferentially.


Highly stereoselective synthesis of (2'R)-[2'-2H]-deoxyribonucleosides and synthesis of their oligonucleotides. Highly stereoselective synthesis of (2'R)-[2'-2H]-2'-deoxyribonucleosides (2'R:2'S = > 99:1) were accomplished by treating 2'-bromo-3',5'-O-TPDS-2'-deoxyribonucleosides with tributyltin deuteride at lower temperatures such as -60 degrees C in the presence of triethylborane. Moreover, synthesis of some oligodeoxyribonucleosides involving them will be described.


High pressure mediated Diels-Alder reaction using 2-iminomalonates as dienophiles: novel synthesis of carbocyclic nucleosides. 9-[c-4-(1,2-Dihydroxyethyl)cyclopent-2-en-r-1-yl]-9H-adenine , an isomer of BCA, has been synthesized from Diels-Alder adducts prepared by the reaction of 2-iminomalonates with cyclopentadiene under high pressure. The synthetic method involves a novel C-N bond cleavage reaction by NaBH4-K2CO3 (RRA reaction conditions) and has wide applicability for the synthesis of various carbocyclic nucleosides.


Synthesis of 3'-substituted-2',3'-deoxy-2'-methylidenepyrimidine nucleosides. 2'-deoxy-2'-methylideneuridine derivative 9 was converted into 2',3'-didehydro-2',3'-dideoxy-2'-phenyl-selenomethyl derivative 16, which was treated with NCS and tert-butyl carbamate to afford 3'-amino derivative 18 via a [2,3]-sigmatropic rearrangement. Treatment of 9 with DAST gave a mixture of 2',3'-didehydro-2', 3'-dideoxy-2'-fluoromethyl derivative 19 and 3'-"up"-fluoro-2'-methylidene derivative 20 in a ratio of 1.5 : 1. On the other hand, when 12 was treated with DAST, 19 and 3'-"down"-fluoro-2'-methylidene derivative 21 were obtained in a ratio of 1 : 1.6. These nucleosides were converted into the corresponding cytidine derivatives 4, 6, and 8, respectively. The reaction mechanisms as well as biological activity of these compounds will also be discussed.


A simple and convenient method for acyclonucleoside synthesis. Introduction of acyclic chain for synthesis of acyclonucleoside derivatives was achieved in a simple and convenient way. Silylated pyrimidine or purine bases were treated with 1,3-dioxolane, trimethyl chlorosilane and metal iodide, such as KI and NaI, all together at room temperature. By this method, 2-thiopyrimidine derivatives were also obtained in good yield, using 2 molecular equivalents of 1,3-dioxolane.


Alkylation reaction of phosphorus oxyacids. Alkylation reactions of phosphorus oxyacids (i.e. phosphoric acid, phosphorous acid, hypophosphorous acid) were examined by means of several methods for the synthesis of the corresponding esters. These reactions were applied to the synthesis of oligonucleotide having phosphotriester bond or phosphonate diester bond.


Synthesis of sialic acid-containing nucleotide sugars: CMP-sialic acid analogs. Syntheses of some sialic acid-containing nucleotide sugars are reported. The reaction of methyl[(2-hydroxy)ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto -2- nonulopyranosid]onate (4) with various fully protected hydrogen phosphonates of nucleotides (5a-c) in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl), gave, after oxidation and deprotection, the corresponding sialic acid-containing nucleotide sugar analogs (8a-c).


Syntheses of oligonucleotide derivatives with P(V) porphyrin and their properties. Two types of oligonucleotide derivatives which are substituted by P(V) porphyrin at the phosphorus atom of an internucleotidic linkage and at the 5'-terminal internucleotidic linkage via a spacer were synthesized (Fig. 1), and hybridization capabilities of them with complementary oligonucleotides were evaluated. A novel method for a sensing of oligonucleotide by the fluorescence quenching via photo-induced electron transfer between the P(V) porphyrin labeled oligonucleotide and pyrene-labeled one on the oligonucleotide template is reported.


Fluorescent dye phosphoramidite labelling of oligonucleotides. A series of fluorescein phosphoramidites (FAM) have been synthesized for use on automated DNA synthesizers. After coupling of the FAM reagents to the 5' hydroxyl of the oligonucleotide on the DNA synthesizer, the excess reagent is removed by washing the solid support. The dye, and its linkage to the oligonucleotide, are stable during the conditions of DNA synthesis and cleavage/deprotection conditions. Purification is attained with the OPC (Oligonucleotide Purification Cartridge), a polystyrene based affinity matrix, which selectively retains hydrophobic oligonucleotide conjugates. Analysis by MicroGel capillary electrophoresis effectively separates fluorescent dye labelled oligonucleotides from unlabelled products.


Strength characteristics of glass-ionomer cements. The purpose of this investigation was to compare the compressive and diametral tensile strengths of two silver-reinforced and three conventional glass-ionomer cements of different powder-to-liquid ratios at 1 and 24 hours. ANOVA (P < 0.001) and Tukey's Studentized Multiple Range Test indicated significant differences between the compressive strengths of several of the cements tested (P < 0.05). No significant differences were noted between any of the cements for the diametral tensile strength test (alpha = 0.05).


The effect of eugenol-containing and eugenol-free temporary cements on microleakage in resin bonded restorations. Eugenol is known to have a detrimental effect on both composite resin and dentin bonding agents. The purpose of this in vitro investigation was to compare the microleakage among groups of resin-luted inlays when the cavity preparations were pretreated with a eugenol-containing temporary cement, a eugenol-free temporary cement, or no temporary cement. Class 5 inlay preparations (20 per group) were completed in extracted human molars. Following the fabrication of composite resin inlays, the preparations were filled with either a eugenol-based temporary cement, a eugenol-free temporary cement, or no cement. After removal of the cement from the cavity preparations and application of a dentin bonding agent, the composite inlays were luted with a resin cement, thermocycled, stained, sectioned, and evaluated for microleakage under a stereomicroscope. None of the groups exhibited significant leakage at the enamel margins. Both of the groups treated with temporary cement leaked at the nonenamel margins significantly more than the control (no cement) group. No significant difference in leakage was demonstrated between the groups treated with the eugenol-containing and the eugenol-free temporary cements.


Carbamide peroxide bleaching: effects on enamel surface hardness and bonding. Three 10% carbamide peroxide home bleaching agents were evaluated to determine their effects on tensile bond strength of resin to enamel and enamel surface hardness. Eighty extracted bicuspid crowns were divided into four groups (three bleaching agents and control), and treated with the bleaching agents for five consecutive days. A bonding site on the buccal surface of each crown was etched with phosphoric acid and an orthodontic bracket bonded in place. The specimens were thermocycled and loaded to failure in an Instron Universal Testing Machine. Five hardness specimens per group were measured prebleaching and after five days' exposure. Analysis by one-way ANOVA indicated no significant differences in bond strength between the four groups (P > 0.05). There were also no differences in pre- or postbleaching Knoop hardness values for the four groups (P > 0.05). This study indicated that in short-term regimens 10% carbamide peroxide does not significantly affect enamel surface hardness or bonding ability.


Pediatric pharmacokinetics and therapeutic drug monitoring. Recent advances in pediatric clinical pharmacology have provided a more rational approach to using several medications in children. An increased understanding of the effect of human development, concurrent medications, organ function, and disease states on the absorption, distribution, metabolism, and excretion of drugs has provided a stronger scientific basis for determining drug dosages in children. By measuring drug concentrations and utilizing pharmacokinetic and pharmacodynamic principles, the probability of therapeutic response can be enhanced for a number of medications. Likewise, therapeutic drug monitoring can minimize the risk of adverse effects from many drugs used in children. However, it must be recognized that toxicity can occur in some patients even though plasma drug concentrations are in the therapeutic range; similarly, some patients may not experience a therapeutic effect when plasma drug concentrations are in the same target range. Therefore, achieving the desired plasma concentration of a drug can enhance both the probability of a therapeutic response and diminish the probability of a toxic response. Therapeutic ranges, however, are only intermediate endpoints that must be used in the context of additional criteria to assess the clinical efficacy of any given drug therapy.


The effect of incremental versus bulk fill techniques on the microleakage of composite resin using a glass-ionomer liner. Incremental placement of composite resin has been suggested to reduce microleakage, particularly at the gingival margin of class 5 cervical restorations. It has become clinically advantageous to place a glass-ionomer liner over dentin to further minimize microleakage resulting from a bond between the dentin and glass ionomer, and glass ionomer and resin. The objective of this study was to compare the microleakage behavior of three hybrid composite/bonding agent systems using bulk and incremental filling techniques utilizing a glass-ionomer liner. This was accomplished in vitro using freshly extracted bovine incisors and a Ca45 radioisotope and autoradiography. Sixty bovine incisors were divided into six experimental groups of 10 specimens per group. Class 5 preparations were cut at the cementoenamel junction and restored with the appropriate combination of Herculite XR/Bondlite, P50/Scotchbond 2, or Pertac Hybrid/Pertac Bond. All teeth were lined with the glass ionomer Ketac Bond before the final restoration was placed. The samples were finished and stored for 24 hours in distilled water before thermocycling. The samples were tested for microleakage using a Ca45 radioisotope technique and autoradiography. Incisal (enamel) and gingival (dentin) margins were scored separately for microleakage but grouped for statistical analysis. Results were analyzed using the Kruskal-Wallis H test. Pertac Hybrid exhibited more leakage than Herculite XR or P50. The difference between microleakage of bulk and incremental filling techniques was only significant for P50.


The influence of matrix use on microleakage in Class 5 glass-ionomer restorations. This in vitro study examined the relationship of matrix use to microleakage in class 5 Ketal-Fil glass-ionomer restorations. Class 5 glass-ionomer restorations were placed on the facial and lingual surfaces of 40 extracted human molars. The occlusal margin was located on enamel, and the cervical margin was located on cementum or dentin. Each tooth had one restoration placed with and without the aid of a soft metal matrix. Specimens were thermocycled (1234 cycles, 6 degrees C -60 degrees C, 30-second dwell time) and immersed in 5% methylene blue dye for four hours. The teeth were sectioned occlusogingivally through the center of each restoration, viewed with an optical microscope (X10), and each restoration was scored for dye penetration around the cavity walls. The enamel and cementum margins were scored separately for the extent of marginal leakage. No difference in leakage was found between restorations placed with or without a matrix (P > 0.05). Enamel restorations leaked significantly less than nonenamel margins, regardless of matrix use (P > 0.05).


Effect of admixed indium on the clinical success of amalgam restorations. The purpose of this study was to clinically evaluate two formulations of a dispersed-phase, high-copper dental amalgam alloy (Indisperse), which contained admixed indium. One alloy tested contained 5% indium, and the second alloy contained 10% indium. A similar alloy without indium, Dispersalloy, was also placed for comparison. Over the course of the five-year study, there were no differences clinically or statistically regarding texture and luster. The margins of the restorations containing indium incurred slightly less fracture than the non-indium-containing restorations; however, these differences were not clinically significant. It can be concluded that the admixture of 5-10% indium as well as the increased ratio of eutectic spheres to lathe-cut particles found in the indium alloys enhance the clinical performance of amalgam restorations.