Comprehensive oral X-ray diagnosis: Scanora multimodal radiography. A preliminary description.

Scanora (Soredex, Orion Corporation, Helsinki, Finland) is a multimodal radiography system which utilizes the principles of narrow beam radiography and spiral tomography. The system includes an integrated multifunction X-ray unit, a coordinate system for object localization and a wide selection of dental and maxillofacial imaging problems. All imaging procedures are computer controlled and executed automatically. Clinical experience and preliminary results show that the multimodal imaging system has great potential in the detection of pathological conditions of the maxillofacial region.


The value of magnetic resonance imaging in the assessment of a sublingual epidermoid cyst.

A case is presented demonstrating the value of magnetic resonance imaging (MRI) in the assessment of a midline sublingual epidermoid cyst in a 22-year-old female. The advantages of MRI compared with CT in the evaluation of soft-tissue lesions in the sublingual space are discussed.


Digital subtraction radiography in artificial recurrent caries detection.

The diagnostic accuracy of digital subtraction radiography in detection of artificial recurrent caries lesions was assessed in this project. The use of digital subtraction radiography has been shown to markedly increase the accuracy of the detection of destruction in the periodontal bone, but the method has not been evaluated in secondary caries detection. Defects of three different sizes, simulating recurrent caries, were sequentially prepared in the interproximal cavity preparation margins of 28 teeth. Two composite restorative materials with different radiographic densities were used as posterior restorations, and a radiograph was obtained of each defect size and restorative material. The radiographs were digitized and subtracted from the reference images, and the conventional radiographs and the subtraction images were evaluated by seven observers. The data were analysed with ROC statistics. Subtraction radiography was found to be superior to conventional radiography in recurrent caries detection, mainly by reducing the false-positive diagnoses. The radiopacity of the restorative material had a significant effect on accuracy with conventional but not with subtraction radiography.


Absorbed doses and energy imparted from tomography for dental implant installation. Spiral tomography using the Scanora technique compared with hypocycloidal tomography.

Tomography is sometimes needed to obtain information on the amount of bone available in the maxilla and in the posterior parts of the mandible prior to implant surgery. Both conventional and computed tomography can be employed. Recently a new imaging device, the Scanora, has been introduced which can be used for spiral tomography. The aim of this study was to compare absorbed doses and energy imparted from this new unit with those from conventional hypocycloidal tomography using the Philips Universal Polytome. A multi-film cassette with five pairs of calcium tungstate screens was used in the latter while a single film technique was used with the Scanora. The absorbed dose measurements were made on an anthropomorphic phantom. Most absorbed doses were found to be below 0.2 mGy except those to the major salivary glands. The absorbed doses with the Scanora were higher than with the Polytome. The beam direction and shorter focus-object distance in the Scanora influenced the absorbed dose distribution. The energy imparted was found to be low for both techniques, 1.8-1.9 mJ with the Scanora for both jaws, and for hypocycloidal tomography 0.78 mJ in the maxilla and 1.3 mJ in the mandible.


Orbital volume measured by a low-dose CT scanning technique.

A method for measuring orbital volume using low-dose CT with contiguous 3 mm transaxial sections is described. The accuracy of the method is 1.6%, as demonstrated by comparing CT volume measurements with those derived directly from alginate impressions and on repeat scanning the precision of the measurement was judged as 1.3%. Within the same individual, the right and left orbital volumes were observed to be within 0.6 cm3 (s.d. +/- 0.33 cm3) of each other. This study demonstrates that low-dose CT scanning is a practical method of determining orbital volume and could be used to advantage in the management of traumatic enophthalmos and blow-out fractures of the orbit.


Effects of additional filtration on image quality in dental radiography: comparison of niobium with aluminium.

Niobium filtration was compared with additional aluminium filtration with respect to the image quality of dental radiographs. An aluminium phantom was constructed which produced densities similar to those found in periapical regions and in enamel. Eight beam qualities were investigated and objective evaluation of the films was made by 20 observers with respect to any effects on radiographic contrast and successful identification of a pattern of holes in the phantom. Radiographic contrast decreased linearly as the half-value layer increased (r2 = 0.960, P < 0.0005), confirming the negative relationship between contrast and half-value layer as previously shown. No specific effect of niobium filtration on radiographic contrast was demonstrated. The number of successful identifications decreased with increasing half-value layer. A regression analysis in the total and dark zones yielded r2 values of 0.937 and 0.943 (P < 0.0005). Similar analyses for both the light and overall zones showed weaker relationships (r2 = 0.734 and 0.742: P = 0.007 and 0.006 respectively). Niobium reduces successful identification to the same extent as equivalent aluminium filtration.


The contribution of frontal tomography to the diagnosis of temporomandibular joint osteoarthritis.

The contribution of frontal tomography to the diagnosis of temporomandibular joint (TMJ) osteoarthritis has been investigated. Ninety-one patients with clinically defined TMJ disorders were examined by simultaneous lateral and frontal TMJ tomograms taken in the intercuspal position. Abnormal findings were observed on frontal tomograms in 31 joints and suspicious findings in 22 joints. On the other hand, there were 41 joints with abnormal findings and 22 joints with suspicious findings on the lateral tomograms. Suspicious or abnormal findings were found on frontal tomograms alone in 14 joints (7.7%) of 14 patients (15.4%). It is concluded that simultaneous lateral and frontal tomography produces a more accurate radiographic diagnosis of TMJ osteoarthritis.


Accuracy of quantification of mandibular condyle displacement in digitally subtracted linear tomograms.

Using a TMJ skeletal phantom, we assessed validity and reliability of digitally subtracted linear tomographic images to quantify condylar position changes. Horizontally corrected frontal and lateral tomographic images were made with the condyle in a 'centred' position and displaced by 1, 2 and 3 mm increments in two of three directions (inferior, and posterior, or lateral). Film images were sequentially subtracted and then randomized so that observers were blinded to the amount and direction of condylar movement. Averages of three measurements of the dark bands representing condylar shifts were made using a digitizing tablet. The study utilized a nested design where the series of film images was subtracted twice and each subtraction was read twice by two examiners. We repeated the study with a second series of films treated in an identical manner. Average differences from expected values were similar for frontal and lateral techniques and all directions and increments of displacement. Typical average difference for any increment was less than 0.1 mm. Standard deviations in measures were similar across techniques and increments and were typically less than 0.1 mm. Significant variation in the absolute value of differences between measured and expected values by direction of condylar displacement was attributed to greater accuracy of registration on the vertical axis for frontal images. A significant source of variation in the study design element 'film set' was attributed to errors in repositioning the phantom.(ABSTRACT TRUNCATED AT 250 WORDS)


Standardized lateral oblique projection of the mandible for digital subtraction radiography.

A special apparatus was developed for standardization of the lateral oblique projection of the mandible (LOPM) in order to apply the digital subtraction method. Geometric reproducibility was examined over an 8-week period using a dry skull. Angulation errors were determined by the relation to image noise in the subtraction image. It was found that the relation between image noise and angulation error was approximated by a second-order polynomial. Geometric reproducibility was within 1 degree over the experimental period. These results suggest that this standardized LOPM is satisfactory for clinical application of digital subtraction. Two cases are presented where this technique was used to demonstrate longitudinal changes in bone density.


Localization of a displaced root fragment by stereoscopy and pluridirecitonal tomography.

The case reported describes two methods used with the Scanora integrated imaging system to locate a root fragment thought to be displaced into the maxillary sinus--rotational narrow beam stereoscopic scanning and wide angle cross-sectional pluridirectional tomography. While the stereo scans located the fragment within the sinus, a more accurate determination of its site, in relation to an object of known position, was obtained from the tomograms.


1992 assessment of radiation risk from dental radiography.

Recent studies suggest that the lifetime cancer risks from exposure to low levels of ionizing radiation may be greater than previously estimated. This review first summarizes the findings of these studies as they pertain to dental radiology, then uses their concepts in combination with dosimetry from the dental literature to estimate the radiation risk from dental radiology. Estimation of risk from groups of exposed individuals requires use of mathematical models that fit the epidemiological data. The ICRP estimates that a single brief whole-body exposure of 1 Gy to 10,000 people results in about 500 additional cancer deaths over the lifetime of the exposed individuals, assuming a dose rate effectiveness factor of 2 for cancers other than leukaemia. Leukaemias are seen as a wave from 5 to 30 years following exposure. Cancers other than leukaemia typically start to appear about 10 years following exposure and remain in excess for as long as most exposed populations are followed, presumably for the lifetime of the exposed individuals. The gonadal dose is so small from dental radiography that the risk of heritable defects is negligible in comparison with the somatic risk. The dental literature contains several studies reporting sufficient dosimetric data for radiosensitive sites in the head and neck to allow estimation of the risk of fatal cancers from intra-oral and panoramic radiography. The highest estimated risks (using the ICRP data) are for leukaemia (bone marrow), thyroid and bone surface cancer. The total risk is estimated to be 2.5 fatal malignancies per 10(6) full-mouth examinations made with D-speed film and round collimation.(ABSTRACT TRUNCATED AT 250 WORDS)


Three-dimensional reconstruction of magnetic resonance images of the temporomandibular joint by I-DEAS.

Evaluation of the temporomandibular joint has been limited by the inability of current technology to image complex morphology and motion in three dimensions. An engineering design program, I-DEAS, has been used to construct solid models from magnetic resonance images. A dried skull with an acrylic resin temporomandibular disc replica, immersed in water, provided sagittal and coronal MR images. Linear dimensions and disc volumes obtained from the models were compared with the original and found to be consistent, within the limits imposed by the slice thickness. We have applied the method to the living joint in an asymptomatic volunteer, and report our initial experience in demonstrating the spatial relationships and motion of the joint components.


A comparison of conventional intra-oral radiography and computer imaging techniques for the detection of proximal surface dental caries.

The detection of proximal surface caries by the visual interpretation of bitewing radiographs is known to be relatively inaccurate. The present study was designed to examine whether computer image processing could improve the diagnostic accuracy. A computer-aided, software-driven, TV-based system was used to digitize conventional radiographs and digitally process the images using histogram equalization and grey-scale inversion to enhance the images. The computer-enhanced images were compared with conventional intra-oral radiographs for the detection of proximal surface caries using receiver operating characteristic analysis. The results indicate that the digital image processing techniques used did not improve the diagnostic accuracy of dental radiographs. No significant difference in diagnostic accuracy could be detected between the non-enhanced digital images and conventional film-based images for the detection of proximal surface caries.


Individualized periapical radiography determined by clinical and panoramic examination.

The efficiency of panoramic radiography compared with full-mouth periapical examination is an unresolved problem. The diagnostic yield of periapical lesions when the clinical signs and symptoms and the findings from a panoramic radiograph served as the basis for an individualized periapical radiographic examination was studied. Two hundred patients were examined clinically and radiographically. The periapical status was assessed step by step with access to increasing numbers of radiographs. For the clinical examination, the sensitivity was 0.24, the positive predictive value 0.62, the specificity 0.98, the negative predictive value 0.90 and the likelihood ratio for the positive test result 12. For radiographs indicated by the clinical examination plus the panoramic radiograph and selected periapical radiographs, both the sensitivity and the positive predictive value were 0.91, the specificity and the negative predictive value 0.99 each and the likelihood ratio was 91. False findings were twice as frequent in the upper as in the lower arch and particularly found in the incisor and premolar regions. In 30% of the patients no periapical radiograph was needed to supplement the panoramic radiograph. In the other patients, two supplementary periapical radiographs were needed on average. We conclude that the information obtained from the clinical and panoramic examinations supplemented with no more than two periapical radiographs will result in a high diagnostic yield on the periapical status.


Recognizing invariant geometric structure in dental radiographs.

The dental radiograph is a non-invasive tool that is used to view internal structures for the diagnosis of caries, periapical lesions and periodontal disease. The requirement for a standardized image is most prominent in periodontal disease since the diagnosis is best done with a difference radiograph. The difficulty is that exact registration for subtraction requires exact reproduction of imaging geometry. A new model of imaging geometry, based on the correspondence of 3D structures, to describe the radiograph formation process is presented. The experimental results show that 3D measurements can be made in dental radiographs (P < 0.01) with up to 16-mm translation errors and angulation errors of up to 32 degrees.


Cost-effectiveness decision analysis of obtaining periapical radiographs of traumatized maxillary incisors.

A long-term decision analysis approach has been applied to the problem of whether or not periapical radiographs should be taken routinely of patients presenting with simple fractures of otherwise asymptomatic maxillary incisors. Information from the literature concerning the accuracy of cold, hot and electrical tests for pulp vitality, combined with data on the accuracy of radiographic diagnosis of periapical pathology, and therefore vitality, has been used in the analysis of the diagnostic problem structured in the form of a decision tree. By adding information concerning the direct costs of examining and treating patients over a 10-year period to the analysis and varying the likely prevalence of periapical lesions in a three-way sensitivity analysis, the following results were arrived at: (1) if the disease prevalence in teenage patients is < 5%, it is economically justifiable to make a visual inspection only and then to proceed with simple restorative treatment; (2) if there is evidence from the clinical examination or patient history that the prevalence of pulp necrosis is between 5% and 50% then, in our case, radiographs were the most appropriate single diagnostic test.


Digital dental image processing of alveolar bone: Macintosh II personal computer software.

Dental radiographs are amenable to digital image processing and analysis for both subjective interpretation and quantification of scene features. Four general-purpose image processing programs for the Apple Macintosh II computer (NIH Image, Enhance, IP Lab and DIP Station) were applied to digital analysis of dental radiographs. These programs can manipulate and quantitatively analyse the gray scale and spatial data of dental images and improve their subjective quality. They can interactively make geometric measurements of features. The programs were evaluated for their versatility and applicability in the quantification of alveolar bone. Overall, NIH Image was judged most suitable for current dental imaging needs. Each of the other programs offered unique features that may be valuable in specific applications.


Iodothyronine 5'-deiodinating activity in the pineal gland.

1. The presence of an iodothyronine 5'-deiodinating activity has been described in the pineal gland of various rodents, and it has been identified as a type II 5'-deiodinase isoenzyme since it is relatively insensitive to inhibition by propylthiouracil and its activity increases during hypothyroidism. 2. 5'-Deiodinase activity in the rat pineal gland follows a nyctohemeral profile, exhibiting basal values during the day and maximal values at night. The nocturnal increase is dependent on the noradrenergic input from the superior cervical ganglia, and both in vivo and in vitro studies show that beta-adrenergic receptors are primarily involved in the activation of the enzyme. 3. Day-night differences in rat pineal 5'-deiodinase activity are found beginning at 2 weeks of age, with rhythms increasing in amplitude until maximal differences are reached in adult animals. During the maturation of the rhythm, changes in regulation of enzyme activation are observed. Thus, during the first 2-3 weeks of age, alpha-adrenergic receptors appear to be as important as beta-adrenergic receptors in regulating the deiodinating activity of the pineal. However, in adults, no role of alpha-adrenergic receptors has been described. 4. Although regulation of 5'-deiodinase activity in the pineal gland is well established, few data are available concerning the physiological significance of the enzyme in the gland. Of the studies that have been performed, those attempting to demonstrate a relationship between pineal 5'-deiodinase activity and other pineal rhythms, e.g. those of melatonin production and N-acetyltransferase activity, indicates that the latter rhythms do not rely on the cyclic production of T3. The alternate possibility that the 5'D rhythm depends on the cyclic production of melatonin remains to be examined.


31P NMR of Mg-ATP in dilute solutions: complexation and exchange.

1. Monovalent-cation [(CH3)4N+, K(I), Na(I)] ATP, 1 mM in nucleotide, in aqueous solutions at pH 7.2, 24 degrees C, generates 2 different 31P NMR spectra, depending upon the salt content of the solution. At salt concentrations below 10 mM, the 31P NMR signals are chemically-shifted upfield (Na salt: alpha, -11.44 delta; beta, -22.91 delta; gamma, -8.36 delta) and the beta- and gamma-groups are broadened (at half-height: alpha, 3.5 Hz; beta, 9.6 Hz; gamma, 69 Hz). Above 10 mM salt, the signals are shifted downfield and are narrow (Na salt: alpha, -11.09 delta, 1.9 Hz; beta, -21.75 delta, 3.3 Hz; gamma, -6.30 delta, 3.9 Hz). 2. The Na-Mg-ATP complex, corresponding to the composition Na6Mg1ATP2, yields a single set of 31P resonances at concentrations of nucleotide of 100 mM, that upon dilution to 0.2 mM, resolve into 2 sets of ATP resonances characterized by low-field and high-field beta- and gamma-group resonance pairs. This set of ATP resonances, in contrast to the resonance set at 100 mM ATP, are broad (100 mM in ATP: alpha, -10.7 delta, 3.7 Hz; beta, -20.1 delta, 15 Hz; gamma, -5.7 delta, 7.3 Hz. 0.2 mM in ATP: alpha, -10.7 delta, 47 Hz; beta, -18.8 and -21.6 delta, 316 and 274 Hz; gamma, -5.5 and -8.7 delta, 460 and 374 Hz). 3. This new data, in combination with data derived from a survey of metal-ion-ATP studies, are interpreted in terms of ATP dimers, incorporating 2 molecules of ATP and 2 metal cations, that exist in water under the physiological conditions of neutral pH, high salt content [135 mM K(I)] and ATP concentrations in the range of 3 mM. 4. A compilation of 31P in vivo and ex vivo data compared to a reference Mg-ATP chemical shift vs Mg/ATP ratio plot indicates that ATP is not fully Mg-saturated in living systems and that 41% exists as the Mg(ATP)2 complex.


Purification of lysosomal arylsulfatase B from rat liver and its reactivity with lectins in affinity immunoelectrophoresis.

1. Arylsulfatase B (ASB) from lysosomal fraction of rat liver were isolated and purified 260-fold with a recovery of about 5%. 2. The enzyme in gradient PAGE 4-30% followed by immunoelectrophoresis migrated as a single peak of M(r) 84,000. The pI, measured by isoelectrofocusing in agarose followed by immunoelectrophoresis, was equal to 6.7. 3. ASB reacted with Con A, LCA, PSA, LTL, WGA, RCAI and did not react with PHA, SBA, HPA, CAA and PAL in crossed affino-immunoelectrophoresis or rocket immunoelectrophoresis. These results permit of preliminary elucidation of ASB glycan structure.


Isolation and characterization of bovine gingival proteoglycans versican and decorin.

1. We have isolated, chemically and immunologically characterized versican and decorin from bovine gingiva. 2. Versican was of large molecular weight and the molecular size of the core protein was estimated to be greater than 200 kDa. 3. The glycosaminoglycan chains were susceptible to chondroitinase ABC and N-linked oligosaccharides were present on the protein core of the molecule. 4. Immunological studies provided evidence that a hyaluronic acid binding region was present in the core protein of versican. 5. The overall structure was similar to that of versican isolated from bovine sclera. 6. Decorin had a molecular weight of 102 kDa and its glycosaminoglycan chain was completely digested by specific glycosidases. 7. The partially deglycosylated core protein had a molecular weight of 55 kDa and N-linked oligosaccharides were present on the molecule.


Use of an immobilized human endogenous lectin for the purification of complementary ligands.

1. A galactoside-specific endogenous lectin isolated from human brain was covalently immobilized on divinylsulfone-activated agarose. This highly selective affinity adsorbent proved to be useful in purifying soluble protein ligands. 2. The maximum binding capacity of the adsorbent for complementary proteins was calculated to be 618 micrograms per g of gel (wet resin). 3. Sequential elutions using 0.1 M lactose, 0.3 M lactose and 0.5 M NaCl, and competition assays using incorporation in the presence 0.1 M lactose revealed differences in lectin-ligand interactions.


Impaired immune responses in diabetes mellitus: analysis of the factors and mechanisms involved. Relevance to the increased susceptibility of diabetic patients to specific infections.

The reasons why diabetic patients present with an increased susceptibility to frequent and protracted infections remain unclear. The virtual absence of epidemiological studies of the independent risk factors involved contrasts with the multitude of in vitro models focused on the metabolism and function of immune cells from diabetic patients. This review analyzes some of these models and their clinical relevance. The different levels of diabetes pathogenesis: genetic (Type 1), autoimmune (Type 1) and metabolic (Type 1 and Type 2) are responsible for immune abnormalities demonstrated in in vitro models. The participation of genetic and autoimmune factors has been mainly characterized on T lymphocyte function. The B8 DR3 haplotype is associated with several minor immunologic abnormalities in vitro. However, the high frequency of this haplotype in healthy individuals argues against its involvement in significant defects of antimicrobial immunity. Genetic deficiency of C4, present in 25% of Type 1 diabetic patients could, on the other hand, be responsible for opsonization defects against encapsulated pathogens. Several immunological abnormalities related to the autoimmune process preceding the onset of Type 1 diabetes mellitus, such as the depletion of memory CD4+ cells and the defective natural killer activity could transiently impair host defences against viral diseases. Several in vitro functional defects of the immune system have been correlated with the metabolic control of diabetic patients. This suggests the involvement of insulinopenia in some of the abnormalities observed. Insulinopenia-induced enzymatic defects have often been proposed to inhibit energy-requiring functions of phagocytes and lymphocytes. However, the relevance of this mechanism could be confined to patients with extremely severe metabolic abnormalities. The importance of systemic consequences of insulinopenia such as hyperglycaemia and ketosis has also been addressed. Usually, the defects induced in vitro by these factors are slight and require supraphysiologic concentrations of glucose or ketone bodies. Recent studies have shown abnormalities of signal transduction mechanisms in which insulinopenia itself and other factors such as circulating immune complexes could be involved. Despite numerous controversies, many in vitro studies of the immune cells of diabetic patients have demonstrated significant defects which bear quantitative similarities with abnormalities described in other immunodeficiency syndromes. Furthermore, several mechanisms have been proposed to link the different defects observed with the specific infections encountered in diabetic patients.


Metabolism of very low density lipoproteins in the pig. An in vivo study.

1. The metabolism of apolipoprotein B (apoB) was investigated in pigs injected with [125I]very low density lipoproteins (VLDL) to determine to which extent the two distinct low density lipoprotein subclasses (LDL1 and LDL2) derive from VLDL. 2. The lipoproteins were isolated by density gradient ultracentrifugation and the transfer of radioactivity from VLDL into LDL1 and LDL2 apoB was measured. 3. Only a minor portion of VLDL apoB was converted to LDL1 (7.7 +/- 3.2%) and LDL2 (3.6 +/- 1.5%), respectively. Thus, we conclude that the major portion of LDL, especially LDL2, is synthesized independently from VLDL catabolism.


Increased muscular phospholipase A2 activity in diabetic rats.

Anomalies in prostaglandin (PG) synthesis have been suggested in both experimental and human diabetes mellitus; increased levels of plasma and tissue eicosanoids has been recently reported by several investigators. One step in prostaglandin synthesis is the enzymatic hydrolysis of membrane phospholipids by Phospholipase A2 (PLA2). Nevertheless the alternative pathway involving Phospholipase C must be considered. An evaluation of PLA2 activity is therefore a useful method for studying prostaglandin synthesis in the peripheral target tissues of insulin activity. We studied PLA2 activity in normal and diabetic rat muscle. Streptozotocin-induced diabetic rats showed significantly higher muscular PLA2 activity when compared with controls (3.04 x 10(-2) +/- 0.50 x 10(-2) versus 1.34 x 10(-2) +/- 0.35 x 10(-2) arachidonic acid pMol.mg protein-1.min-1 (p less than 0.01). This effect was not observed in diabetic animals successfully treated with insulin (1.78 x 10(-2) +/- 0.5 x 10(-2) versus 1.34 x 10(-2) +/- 0.35 x 10(-2) arachidonic acid pMol.mg protein-1.min-1), and a significant correlation was found between blood glucose and muscular PLA2 activity (r = 0.42; p less than 0.05). Our results clearly show that in streptozotocin-induced diabetic rats muscular PLA2 activity is significantly higher. The relationship between blood glucose levels and muscular PLA2 activity and the decrease of PLA2 activity after insulin treatment suggest that these changes may be related to a defect in insulin effect.


Pregnancy modulates the expression of beta-N-acetylhexosaminidase in rat serum and tissues.

1. beta-N-Acetylhexosaminidases in maternal rat serum were separated by DEAE-cellulose chromatography and compared with those of adult rat serum. 2. In pregnant serum there is an increase of the isoenzymes which are entirely composed of beta-subunits (B and intermediate forms). 3. These alterations could be compared to those already described in human pregnancy. 4. The levels of beta-N-acetylhexosaminidase and the relative expression of alpha- and beta-subunits in normal and pregnant serum correlate with the above isoenzyme expression. 5. The increase of B and intermediate forms as well as the increase of specific activity during pregnancy was not peculiar to maternal serum but was also demonstrated in several foetal tissues and in maternal tissues, in which cases the beta-N-acetylhexosaminidase isoenzyme patterns closely resemble the foetal ones rather than those of the adult rat tissues. 6. These analogies strongly suggest that the expression of beta-subunit of beta-N-acetylhexosaminidase is regulated by hormones or other factors related to pregnancy.


[Known diabetes mellitus in Algeria: frequency and sequelae].

A sampling survey was carried out in Algiers in order to assess the frequency of the previously known diabetes mellitus and to appraise the handicap resulting from this disease. This study was undertaken among 1,302 households, i.e., 9,384 inhabitants. A questionnaire was used to identify people with diabetes. The overall prevalence of diabetes was 21.7 per thousand inhabitants while the annual incidence was estimated as 2.0 per thousand. 18.1% of previously known diabetics were insulin-treated. Diabetes became strikingly more prevalent with advancing age, being over 140 per thousand among women after 60 years. There was no significant difference in the prevalence rates of diabetes among the different social levels. Diabetes was scarcely revealed by a systematic examination (20%). More than one third of the diabetic patients were without any handicap. The most serious handicaps were notably observed among old people. Significant differences between the mean ridits of handicap were observed only when people over 60 were compared to the other age groups for physical independence, mobility and occupation.


Neuropathy target esterase is not reduced in neural tissues of diabetic rats.

Marked (greater than 70%) reduction of the neuropathy target enzyme (NTE) shortly after exposure to organophosphorus compounds heralds the onset of delayed neuropathic damage in animals and humans. One previous study reported that lymphocyte NTE from diabetic patients was depressed by greater than 70%; such a reduction was considered to be a biological marker of diabetic polyneuropathy. To ascertain whether NTE from target tissues might be involved in diabetic neuropathy, we measured NTE activity in the brain, spinal cord and peripheral nerves of streptozotocin-diabetic rats. No reduction in NTE activity was detected in these neural specimens. Therefore, it is concluded that NTE is not involved in diabetic nerve damage and that the meaning of low NTE activity in peripheral lymphocytes of diabetic patients remains unclear.


Role of prostaglandin E2 (PGE2) in the growth hormone releasing hormone action on growth hormone, insulin and C-peptide in normal men.

Fourteen healthy men were placed in two treatment groups. 1. Seven men received growth releasing hormone (1 microgram/kg BW) intravenously during either saline or prostaglandin E2 (10 micrograms/min) infusion which were started 60 minutes before testing. Growth hormone response to growth releasing hormone was greater during prostaglandin E2 infusion compared to the control study. These data suggest that exogenous prostaglandin E2 positively modulates growth hormone response to growth releasing hormone. In the second group consisting also of 7 men a GRH test was performed before and after 3 days administration of meclofenamate. Meclofenamate significantly reduced plasma levels of stable metabolites of prostaglandin E2 as well as the growth hormone to growth releasing hormone, suggesting that the GRH effect on GH was at least partly under prostaglandin E2 control. GRH significantly reduced serum insulin in the two treatment groups. PGE2 infusion or meclofenamate did not change insulin response to GRH. Serum C-peptide and blood glucose did not change after GRH injection during saline or PGE2 infusions.


Inhibition by aluminum ion of NAD- and NADP-dependent isocitrate dehydrogenases from yeast.

1. NADP-dependent isocitrate dehydrogenase from yeast was potently inhibited by aluminum ion competitively with respect to the substrate isocitrate, and noncompetitively with the other substrate NADP. Ki value was determined to be 0.43 microM. 2. Aluminum ion acted as only a weak allosteric inhibitor of yeast NAD-dependent isocitrate dehydrogenase toward isocitrate, and as a noncompetitive inhibitor toward NAD. 3. Inhibition by aluminum ion of NADP- and NAD-isocitrate dehydrogenases can reduce the aerobic energy production in yeast, and may contribute to the biological toxicity of aluminum in ecosystems and human life.


Lipoxygenase activity in the brain regions of young chicks: isolation and some properties.

1. The lipoxygenase (LOX) oxygenation pathway of arachidonic acid was investigated in the cerebellum and cerebral hemispheres of young chicks. 2. Lipoxygenase products consisted mainly of 15-hydroxyeicosatetraenoic acid (15-HETE), accompanied by the 15-hydroperoxy analog (15-HPETE) and the 5-HETE product. 3. The yield of 15-HETE was 3 times greater in the cerebellar system than in the cerebrum. 4. PLA2 activity of the cerebellum was twice that of the cerebrum. 5. Affinity chromatography revealed 2 brain fractions with LOX activity which were assayed with either linoleic or arachidonic acid as substrate. 6. The fraction eluted with 0.2 M sodium acetate pH 5.0, produced a higher yield and enrichment of LOX activity than the eluate obtained with 0.1 M Tris-HCl buffer (pH 8.0). 7. A considerably higher yield and enrichment of the enzyme was achieved when the starting material was the cerebellum, compared to the cerebrum. 8. The optimal pH for both purified fractions from cerebrum and cerebellum was 6.5, with either linoleic or arachidonic acid as substrate. 9. The cerebral LOX yielded Michaelis-Menten kinetics when linoleic acid was the substrate, while the corresponding plots for the cerebellar enzyme were sigmoidal. 10. Arachidonic acid as substrate produced sigmoidal plots, except at pH 5.0, where Michaelis-Menten kinetics were observed. 11. These results and the elevated activities of PLA2 and 15-LOX could be significant in relation to the special vulnerability of the cerebellum in chick nutritional encephalomalacia.


Comparison of the effects of bezafibrate and acipimox on the lipid pattern and plasma fibrinogen in hyperlipidaemic type 2 (non-insulin-dependent) diabetic patients.

Correction of cardiovascular risk factors is of particular significance in a high-risk population, such as that of diabetic patients. This paper reports the effects of one-month administration of 400 mg/day Bezafibrate (BZF), followed by a two-month wash-out and one-month administration of 500 mg/day Acipimox (APX) or vice versa in a random order in 16 Type 2 diabetic patients with diet-resistant hyperlipidaemia and in good metabolic control (HbA1c less than 8%), on plasma fibrinogen and on their lipid pattern. Metabolic control displayed a nonsignificant improvement (HbA1c) during both treatments (stable body weight). Both BZF and APX produced a 14% decrease in total CHOL (p less than 0.01), whereas BZF was more effective in reducing triglycerides (tg) (-37% vs -15%). The marked BZF-induced Tg reduction was associated with a proportional decrease in Apo B, while an increase in total HDL-, HDL2 and HDL3-CHOL, together with a significant increase in Apo AI, was observed. APX treatment resulted in a HDL2-CHOL increase only (+29%). Both drugs reduced VLDL-CHOL (BZF -37%; APX -15%) and VLDL-Tg (-56% and -34%). In BZF treated patients Apo CIII fell indicating a possible reduction of specific inhibition of lipoprotein lipase activity, while APX affected both Apo CII (+23%) and Apo CIII (-26%) and led to a 62% Apo CII/CIII ratio increase. BZF alone led to a significant 25% decrease in plasma fibrinogen (from 415 +/- 14.3 to 312.1 +/- 18.1 SEM mg/dl, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Acid phosphatases in mammalian tissues. Evidence for the existence of a 57 kDa Zn(2+)-dependent acid phosphatase form.

1. A comparative study of multiple forms of acid phosphatase (AcPase) in various organs of mammals was carried out. 2. These studies indicated that the high-molecular weight AcPase is preferentially expressed by tissues which undergo cell proliferation such as epithelial tissues; on the contrary, the low-molecular weight enzyme seems to be characteristic of highly differentiated tissues such as nervous, muscle and blood erythrocytes. 3. The existence of a new AcPase activated by Zn2+ ions was observed in all tissues studied with the exception of erythrocytes. 4. The enzyme shows a molecular weight of 57 kDa, is insensitive to NaF, hydrolyzes p-nitro-phenylphosphate and o-c-phenylphosphate; ATP, a-naphthyl-phosphate and beta-glycerolphosphate are also dephosphorylated.


Modulation of ribonuclease P expression in Escherichia coli by polyamines.

1. The presence of polyamines in the growth medium of Escherichia coli can modulate the activity of the RNA-processing enzyme, ribonucleoprotein ribonuclease P (RNase P), by altering the expression of the rnpA and rnpB genes, which encode its C5 protein and M1 RNA subunits, respectively. 2. Following growth in the presence of 1 mM spermidine the levels of C5 protein mRNA and catalytic M1 RNA were significantly elevated in the wild type E. coli K-12 strain MG1655. 3. The rnpA mRNA, together with the ribosomal protein L34 (rpmH) mRNA, was found to constitute a dicistronic rpmH-rnpA message whose half-life did not change upon Escherichia coli growth in the presence of spermidine. 4. This suggests that the spermidine effect is on the transcriptional level. 5. Increased expression of the rnpA and rnpB genes was reflected in the activity of RNase P, which almost doubled. 6. These results identify yet another component of the protein synthetic machinery which is specifically affected by polyamines.


Rat guanidinoacetate methyltransferase: mutation of amino acids within a common sequence motif of mammalian methyltransferase does not affect catalytic activity but alters proteolytic susceptibility.

1. Manual alignment of amino acid sequences of mammalian S-adenosylmethionine-dependent methyltransferases of known sequence revealed the presence of 2 homologous regions. 2. The sequence of the region at the C-terminal side is unique to mammalian methyltransferases, and in guanidinoacetate methyltransferase this sequence occurs at residues 159-165. 3. Mutagenesis of 5 conserved residues in this sequence did not affect the catalytic activity but altered tryptic susceptibility at Arg20.


The control of protein degradation in monolayer cultures of cat hepatocytes.

1. Isolated cat hepatocytes were established in monolayer culture, cell proteins labelled with tritiated leucine and the effects of amino acids and hormones on the regulation of intracellular protein breakdown were studied. 2. Mixtures of essential and non-essential amino acids inhibited the breakdown of long-lived protein, but when tested individually, amino acids except for tryptophan were ineffective. 3. The rate of breakdown of short-lived protein was not regulated by amino acids or hormones, a finding which was similar to that in rat liver cells. 4. The known stimulatory hormones of proteolysis in rat liver such as glucagon, dexamethasone and corticosteroids failed to enhance protein degradation in cat liver cells. 5. These results support the contention that the control of protein degradation in the cat is different to that in the rat and these differences may reflect the unusual protein metabolism of the cat.


Sequence of insulin effects on cytoskeletal and cytosolic phosphofructokinase, mitochondrial hexokinase, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate levels, and the antagonistic action of calmodulin inhibitors, in diaphragm muscle.

1. Time-curves of insulin effects on energy-producing systems in different cellular compartments of rat diaphragm muscle have revealed: (a) a rapid (within minutes) and transient stimulatory effect of insulin on cytoskeletal phosphofructokinase and aldolase and mitochondrial hexokinase. (b) A slower and consistent stimulatory effect on glucose 1,6-bisphosphate level, with concomitant gradual activation of cytosolic phosphofructokinase. Fructose 2,6-bisphosphate levels were not changed by insulin. (c) Lactate concentration correlated with the stimulation of cytoskeletal and cytosolic glycolysis. 2. Calmodulin antagonists, trifluoperazine or CGS 9343B, prevented all these effects of insulin. 3. These results suggest that cytoskeletal glycolysis and mitochondrial oxidation are the source of ATP for the rapid actions of insulin, whereas cytosolic glycolysis is the source of ATP for the slow actions of insulin. Calmodulin is involved in all these effects of insulin.


Phorbol ester induction of rat hepatic metallothionein in vivo and in vitro.

1. The induction of metallothionein (MT) protein by TPA (O-tetradecanoyl phorbol acetate), a protein kinase C activator, was demonstrated in vivo in rat liver and in vitro in rat hepatocytes in primary culture. In vivo half maximal induction at 25 hr was seen at 26 nmol TPA/kg body wt. Five- to seven-fold inductions were seen in vivo. De novo protein synthesis was required for this induction as demonstrated by cycloheximide inhibition of [35S]cysteine incorporation into MT protein. 2. TPA induction of MT protein in primary cultures of rat hepatocytes reached levels of 2.6-4.1-fold, as assessed by [35S]cysteine incorporation, 1.34-2.20-fold, as assessed by 109Cd binding in a metal displacement/HPLC assay, and 2.5-5-fold, as assessed by 109Cd binding in a metal displacement/Sephadex G-75 Superfine assay. 3. The induction of MT mRNA by TPA was demonstrated in vivo in rat liver and in vitro in 2 rat hepatoma cell lines, EC3 and 2M. MT mRNA was quantitated using dot blot and Northern gel assays. In vivo TPA induced hepatic MT mRNA 2.36-5.88-fold (dot blot) and 7.4-22-fold (Northern gels). In vitro TPA induced MT mRNA 1.71-15.26-fold in EC3 cells and 2.23-8.43-fold in 2M cells. MT mRNA was 0.54 kb, and alpha-tubulin mRNA was 1.62 kb in size on Northern gels. 4. TPA induction of MT protein and mRNA in vivo and in vitro is rapid and persistent and occurs at low concentrations. The 2 rat hepatoma cell lines provide a useful system in which to study MT induction in vitro without confounding secondary effects which can occur in vivo.


The effects of propionate and butyrate on acetate metabolism in rat hepatocytes.

1. Two mM propionate or butyrate inhibited the mitochondrial uptake of acetate by rat hepatocytes. 2. With propionate the inhibition was so strong that the net formation of acetate in the cytoplasm, usually masked by the mitochondrial uptake, appeared directly as a net output of acetate into the medium; showing that this net formation of acetate, reported by [Crabtree B., Gordon M.-J. and Christie S. L. (1990) Biochem. J. 270, 219-225] is not an artefact arising from a misinterpretation of isotopic data. 3. The results also suggest that propionate and butyrate inhibit peroxisomal metabolism.


Hemolysis of phosphatidylcholine-containing erythrocytes by serratamic acid from Serratia marcescens.

1. The hemolysis by serratamic acid, "N-(D-3-hydroxydecanoyl)-L-serine and N-(D-3-hydroxydodecanoyl)-L-serine", was investigated with human and animal erythrocytes using serratamic acid-containing liposomes. 2. The hemolytic activity was found to depend on the incubation temperature and the concentration of the liposomes. 3. The concentration of serratamic acid for 50% hemolysis was 0.17 mM at 37 degrees C for 0.2% human erythrocyte suspension in the liposomes which composed of phosphatidylserine, cholesteryl nervonate and serratamic acid (1:0.50:0.37 by mol). 4. The hemolysis was shown specifically in human, horse and rabbit erythrocytes containing phosphatidylcholine, but not in sheep or bovine erythrocytes lacking phosphatidylcholine. 5. The hemolytic activity was strongly inhibited by the exogenous addition of phosphatidylcholine. It was suggested that the hemolysis by serratamic acid-containing liposomes was specific for phosphatidylcholine-containing erythrocyte membranes.


The effect of L-carnitine on cholesterol metabolism in rat (Rattus bubalus) hepatocyte cells.

1. This paper concerns the study of the effect of L-carnitine on cholesterol metabolism in rat hepatocyte cells BRL-3A. In this research the binding of [125I]human low density lipoprotein (LDL) to BRL-3A cells and 3-hydroxy 3-methylglutaryl CoA reductase activity (HMG-CoA reductase activity) after L-carnitine incubation were studied. 2. It was found that L-carnitine is able to increase either the [125I]LDL binding or inhibit the HMG-CoA reductase activity in BRL-3A cells. 3. These results indicate that L-carnitine affects the cholesterol metabolism through an inhibition of HMG-CoA reductase activity that could be responsible for the increased [125I]LDL binding in rat hepatocytes.


Kinetic evidence for the existence of an unstable intermediate in the trinitrophenylation-induced rhodanese inactivation reaction.

1. Rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate, in the presence of n-butylamine in the reaction medium, has been studied by a kinetic analysis of the data, based on the assumption that enzyme inactivation is brought about by direct reaction of this with the modifying agent. 2. Initial reaction rates for rhodanese activity loss were determined by a mathematical analysis of the first three recorded values of rhodanese residual activity. 3. It was found that fractional rhodanese activity values, at infinite reaction time with 2,4,6-trinitrobenzenesulphonate (end-point values), were significantly lower than the values calculated on the assumption of rhodanese inactivation being entirely due to direct trinitrophenylation of enzyme protein. 4. Also, initial enzyme inactivation values were higher in the presence, rather than in the absence, of n-butylamine. 5. These results indicate that 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation, in the presence of n-butylamine in the reaction medium, is due to the generation of a highly reactive, unstable intermediate, probably a free radical species.


Characterization of 130 kDa protein from rat cerebellum synaptosomal membranes phosphorylated by PKC.

1. The effect of endogenous PMA-stimulated phosphorylation of the protein in the molecular weight range of 130 kDa in rat cerebellum synaptosomal membranes was examined. 2. The 50% inhibition of the phosphorylation of 130 kDa protein by 5 microM polymyxin B was observed after 6 min of preincubation. 3. The sensitivity of 130 kDa protein for phosphorylation in the presence of exogenous protein kinase C suggests, that this protein could serve as a physiological substrate of protein kinase C. 4. Partial characterization of 130 kDa protein was performed. Upon incubation with [gamma-32P]ATP the 130 kDa protein formed Ca(2+)-dependent, hydroxylamine-sensitive phosphointermediate, which was inhibited by 50 microM vanadate, but not 0.5 mM vanadyl. 5. One-dimensional peptide mapping by proteolysis of 130 kDa protein with V8 protease was obtained.


In vivo effects of cadmium on rat liver glucocorticoid receptor functional properties.

1. Cadmium (Cd2+) administered in vivo induced a 40% reduction of rat liver glucocorticoid receptor (GR) capacity and inhibition of glucocorticoid-receptor complexes binding to mouse mammary tumor virus (MMTV) DNA fragment containing GR consensus sequence. 2. The effect of Cd2+ on the GR binding activity can be reversed with DTT, suggesting Cd2+ interaction with thiol groups. 3. Cd(2+)-related GR modification seems to be mediated by Cd2+ binding to cytoplasmic components included in the regulation of the receptor function, although the direct binding of the metal to the receptor thiols could not be ruled out.


Purification and characterization of streptolysin O from Streptococcus pyogenes.

1. Streptolysin O, an exotoxin produced by group A beta-hemolytic streptococci, has been purified from Streptococcus pyogenes culture supernatants. 2. The isolation and purification procedure involved ammonium sulphate and polyethylene glycol precipitations, and ion-exchange chromatographies on CM-Sepharose and Mono Q. 3. The proposed procedure introduces two ion-exchange chromatography steps making the purification process simpler and, especially, more reproducible than other described protocols. 4. The purified streptolysin O was hemolytically active, had a specific activity of 415,000 HU/mg, an optimum pH of 7.0, a relative molecular mass of 60,100 and an isoelectric pH of 7.5.


Changes in the properties of honeybee haemolymph alpha-glucosidases following dithiothreitol dissociation of native complexes.

1. The higher relative molecular mass (M(r)) forms of larval honeybee haemolymph alpha-glucosidases are dissociated by dithiothreitol (DTT) into lower M(r) electrophoretic forms, without any important loss of activity. 2. The maximum velocity remains unchanged and the apparent dissociation constant is slightly increased, with Ki approximately equal to 247 mM and I50 approximately equal to 730 mM. 3. By contrast, the major changes affect the Hill coefficient which decreases from 1.0 in controls to 0.7 in presence of 600 mM DTT. 4. In the absence of both DTT and substrate, the major native enzyme form, isolated by gel filtration, spontaneously rearranges to give three additional minor forms, one of lower M(r) and two of higher M(r). 5. These data are consistent with the hypothesis of substrate-directed aggregation of enzyme protomers into functional complexes.


The isolation and partial characterization of catalase and a peroxidase active fraction from human white adipose tissue.

1. A procedure is described for the purification of catalase and a peroxidase active fraction from human white adipose tissue. 2. Gel electrophoresis on SDS-PAGE revealed relative molecular masses of 202,900 and 208,600 for the active catalase and peroxidase molecules respectively (nonreducing conditions), as compared to 56,800 and 49,800 for the monomers under reducing conditions, thus indicating the likelihood of tetramers in the intact state. 3. The two purified enzymes differ with regard to pH optima (5-9 for catalase and 3 for peroxidase), temperature stability (up to 50 degrees C for catalase and 70 degrees C for peroxidase) and Km values towards H2O2 (38.9 mM for catalase and 7.69 mM for peroxidase, which was also active in oxidizing a number of o-dihydricphenols as second substrates). 4. The catalase enzyme showed uncompetitive inhibition by the irreversible inhibitor 3-amino-1,2,4-triazole (AT), Ki = 5.4 mM.


Beta-oxidation as channeled reaction linked to citric acid cycle: evidence from measurements of mitochondrial pyruvate oxidation during fatty acid degradation.

1. The kinetics of mitochondrial mammalian pyruvate dehydrogenase multienzyme complex (PDHC) is studied by the formation of CO2 using tracer amounts of [1-14C]pyruvate. It is found that the Hill plot results in a (pseudo-)cooperativity with a transition of n-1----3 at a pyruvate concentration about Ks. 2. Addition of L-carnitine, octanoate, palmitoyl-CoA or palmitate + L-carnitine + fatty acid-binding protein results in a Hill coefficient of n = 2 following the kinetics of pyruvate oxidation. 3. Addition of fatty acid-binding protein to an assay system oxidizing palmitate in presence of L-carnitine alters the pattern of the kinetics in the Hill plot so that an apparently lower level of L-carnitine is necessary for the reaction course of beta-degradation. 4. It is concluded that beta-degradation is a coordinated, multienzyme-complex based mechanism tightly linked to citric acid cycle and it is proposed that L-carnitine is actively involved into the reaction and not only functioning as carrier-molecule for transmembrane transport.


Physiological state and the sensitivity of liver mitochondrial outer membrane carnitine palmitoyltransferase to malonyl-CoA. Correlations with assay temperature, salt concentration and membrane lipid composition.

1. Liver mitochondrial outer membranes were pre-exposed to media of low (20 mM phosphate) or high salt concentration (20 mM phosphate + 0.3 M KCl) before assay of carnitine palmitoyltransferase (CPT) at 25 degrees C. 2. With membranes from fed rats, exposure to high salt decreased sensitivity of CPT to malonyl-CoA whereas high salt increased sensitivity of CPT to malonyl-CoA in membranes from 48 hr-fasted rats. These changes were paralleled by alterations in the KD for high affinity binding of [14C]malonyl-CoA to outer membranes. 3. Decreasing the CPT assay temperatures from 25 to 10 degrees C caused qualitatively similar changes to those seen on exposure to high salt. 4. The relative content of sphingomyelin was increased 2-fold and 4-fold in liver mitochondrial outer membranes from fasted and diabetic rats respectively. Fasting had no effect on the content of cholesterol whereas diabetes decreased this by a third.


Ribosomes from trichomonad protozoa have prokaryotic characteristics.

1. Ribosomes from cells of the genera Trichomonas and Tritrichomonas have been isolated and characterized. The ribosomes from each organism had a sedimentation coefficient of 70S in calibrated sucrose gradients and the subunits sedimented as 50S and 30S particles under the same conditions. 2. The major ribosomal RNAs from each species were identical in size to prokaryotic ribosomal RNAs when examined by denaturing gel electrophoresis. The ribosomes contained both 5.8S and 5S RNAs. 3. The ribosomal proteins were compared by the methods of two-dimensional gel electrophoresis and reversed phase HPLC. Electrophoresis of the ribosomal proteins in two different gel systems indicated the presence of 56 proteins in T. gallinae, 40 in T. bactrachorum and 45 in the Tritrichomonas sp. The protein molecular mass range was 8.5-40 kDa. 4. The HPLC analysis confirmed the protein number established by the gel methods. 5. Both methods of analysis revealed greater similarities between the ribosomal proteins of the 2 Tritrichomonas sp. than between those of the more distantly related T. gallinae and T. bactrachorum.


Proteolysis at pressure and HPLC peptide mapping of M4-lactate dehydrogenase homologs from marine fishes living at different depths.

1. The effects at 10 degrees C of moderate hydrostatic pressure (136 atm) on trypsinolysis of muscle-type (M4) lactate dehydrogenase homologs (LDH, EC 1.1.1.27, L-lactate:NAD+ oxidoreductase) from shallow- and deep-occurring marine fishes were examined by mapping the partial digests by reverse phase HPLC. 2. Comparison of peptide maps of digests generated at 1 and 136 atm revealed that increased pressure did not expose new cleavage sites in homologs of any of the species; no new peptides were generated. 3. Increased pressure did alter the relative amounts of peptides produced. The net effect of increased pressure was to increase the amount of peptides generated in the shallow-occurring species. For deep-living species pressure did not alter the net amount of peptides produced compared to the 15 min atmospheric pressure samples, although the relative amounts of some of the peptides changed. Incubation at 136 atm for 30 min decreased the net amount of peptides produced. 4. It is suggested that the effects of pressure on trypsinolysis may result from slight conformational changes in the substrate proteins.


Purification and characterization of a glycosylated proline-rich protein from human parotid saliva.

1. A glycosylated proline-rich protein (GPRP) was purified to homogeneity by subjecting parotid saliva to immunoaffinity, cation exchange, affinity and hydrophobic chromatography. 2. The purified GPRP had a molecular weight of 78 kDa as analyzed by SDS-PAGE. 3. The amino acid analysis revealed a preponderance of proline, glycine and glutamic acid/glutamine, which accounted for 77% of the total amino acids. 4. Cysteine, tyrosine or phenylalanine residues were not detected. 5. The glycoprotein contained 34% neutral sugars and the oligosaccharides were rich in mannose and N-acetylglucosamine, indicating that N-linked oligosaccharides were the predominant type of oligosaccharides in the molecule. 6. These observations were confirmed by treatment of the purified glycoprotein with specific N-glycosidase which removed the N-linked oligosaccharides leaving a core protein with an apparent molecular weight of 51 kDa. 7. The isoelectric point of GPRP was approx 7.0 and the molecule was not affected by reduction with 2-mercaptoethanol, indicating that no disulfide linkages were present. 8. The GPRP bound to hydroxyapatite and this binding could be partially inhibited by preincubation of the hydroxyapatite with parotid or submandibular saliva. 9. The purified GPRP also bound to a protein with an apparent molecular weight of 95 kDa present in submandibular saliva.


Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).


Colworth Medal Lecture. Glycosyl-phosphatidylinositol membrane anchors: the tale of a tail.

Over the last few years we have learned of a new type of membrane anchorage for cell-surface proteins, the GPI anchors. We now have a good idea of their structure and biosynthesis, and indications of their specific functions in protozoa and mammals. The widespread distribution of GPI membrane anchors throughout the eukaryotes has led many researchers to work on various aspects of GPI anchors. This has led to the widespread exchange of data, ideas and techniques which has made the field a pleasure to work in.


Catalytic mechanisms and regulation of lignin peroxidase.

Lignin peroxidase (LiP) is a fungal haemoprotein similar to the lignin-synthesizing plant peroxidases, but it has a higher oxidation potential and oxidizes dimethoxylated aromatic compounds to radical cations. It catalyses the degradation of lignin models but in vitro the outcome is net lignin polymerization. LiP oxidizes veratryl alcohol to radical cations which are proposed to act by charge transfer to mediate in the oxidation of lignin. Phenolic compounds are, however, preferentially oxidized, but transiently inactivate the enzyme. Analysis of the catalytic cycle of LiP shows that in the presence of veratryl alcohol the steady-state turnover intermediate is Compound II. We propose that veratryl alcohol is oxidized by the enzyme intermediate Compound I to a radical cation which now participates in charge-transfer reactions with either veratryl alcohol or another reductant, when present. Reduction of Compound II to native state may involve a radical product of veratryl alcohol or radical product of charge transfer. Phenoxy radicals, by contrast, cannot engage in charge-transfer reactions and reaction of Compound II with H2O2 ensues to form the peroxidatically inactive intermediate, Compound III. Regulation of LiP activity by phenolic compounds suggests feedback control, since many of the products of lignin degradation are phenolic. Such control would lower the concentration of phenolics relative to oxygen and favour degradative ring-opening reactions.


A comparison of peroxidase and cytochrome P-450.

A peroxidase has 1-electron oxidation as its characteristic activity, while that of cytochrome P-450 is hydroxylation. Both catalytic cycles involve similar high valency states of iron. However, a peroxidase can only accept electrons at the haem edge, while the substrate for cytochrome P-450 is bound in a precise orientation before the active state is created.


Discovery and characterization of an HIV-1 Tat antagonist.

Ro 5-3335, 7-chloro-5-(2-pyrryl)-3H-1,4-benzo-diazepin-2-(H)-one, has been shown to inhibit gene expression controlled by the human immunodeficiency virus-1 (HIV-1) LTR promoter. The inhibition was specific for the viral transcriptional transactivator Tat. The compound did not inhibit the basal activity of the HIV-1 LTR or the activity of promoters not responsive to Tat. Consistent with its mode of action, Ro 5-3335 inhibited HIV-1 replication (IC50 = 0.1-1 microM) by reducing viral RNA synthesis in acutely, as well as chronically, infected cells in vitro. The compound was active against HIV-1 and HIV-2, and AZT-resistant clinical isolates.


Postprandial lipoprotein metabolism in obese patients with moderate hypertriglyceridaemia: effects of gemfibrozil.

After an oral fat load of 1 g/kg body weight in 10 obese females with hyperlipoproteinaemia type IV, serum triglycerides concentrations were maximal at 4 h with a slight decline at 6 h, whereas serum cholesterol concentrations rose slightly at 4 h and 6 h. After 6 h, concentrations of triglycerides and cholesterol were significantly increased in chylomicrons and very low-density lipoprotein (VLDL), whereas cholesterol concentrations were decreased in high-density lipoprotein 2 (HDL2) plus HDL3. After oral treatment with 450 mg gemfibrozil twice daily for 28 days, triglyceride concentrations were reduced in serum, chylomicrons, VLDL and low-density lipoprotein, and total cholesterol concentrations were reduced in serum, chylomicrons and VLDL, and increased in HDL2 plus HDL3. At 6 h after a fat load following 28 days' gemfibrozil treatment, triglyceride and cholesterol concentrations were reduced in serum, chylomicrons and VLDL when compared with pretreatment results. It is concluded that gemfibrozil is effective in lowering triglycerides and cholesterol, particularly in triglyceride-rich particles, and raising the cholesterol content of HDL2 plus HDL3. After an oral fat load gemfibrozil inhibits the increase in serum cholesterol and partly prevents postprandial hypertriglyceridaemia.


A comparative study of nicardipine and pindolol as second-line treatments in essential hypertension.

A controlled, randomized, single-blind, parallel-group study compared the effects of nicardipine hydrochloride/hydrochlorothiazide (HCTZ) with those of pindolol/HCTZ in treatment of essential hypertension. The study included 43 patients aged 30-64 years with supine diastolic blood pressures between 95 and 125 mmHg at baseline. Patients initially received 50 mg/day HCTZ for 6 weeks and those patients whose diastolic blood pressure remained at or above 90 mmHg at week 6 (n = 29) completed a 6-week comparative phase in which they were given, in addition, either 30 mg nicardipine hydrochloride or 5 mg pindolol three times daily. Nicardipine was more effective than pindolol as a second-line treatment in controlling blood pressure but, because patients who were treated with nicardipine/HCTZ had higher baseline blood pressures, significance was lost when results were adjusted for the baseline blood pressure values. Treatment was described as 'very good' by 71.4% of patients in the nicardipine/HCTZ group and by 53.9% of those in the pindolol/HCTZ group; thus, both second-line antihypertensives were well accepted. Although 45% of patients in of each treatment group reported treatment-related adverse events, none experienced postural hypotension and no adverse event was unexpected.


Natural cartilage polysaccharides for the treatment of sun-damaged skin in females: a double-blind comparison of Vivida and Imedeen.

Evidence is accumulating that cartilage polysaccharides derived from marine fish have a repairing effect on solar elastosis. In a double-blind trial, the efficacy and safety of two commercial preparations, Vivida and Imedeen, were compared in the treatment of sun-damaged skin in women aged 40-60 years. A group of 15 women received 500 mg/day Vivida and another 15 women received 380 mg/day Imedeen orally for 90 days. Subjective assessment revealed statistically significant improvements in skin condition in both treatment groups but Vivida was significantly (P less than 0.01) more effective than Imedeen for all parameters. In the Vivida group, mean epidermal thickness increased from 0.14 to 0.26 mm, dermal thickness from 0.90 to 1.51 mm and the elasticity index from 47% to 71%. In the Imedeen group, epidermal thickness increased from 0.13 to 0.18 mm, dermal thickness from 0.80 to 0.97 mm and the elasticity index from 48% to 56%. After 90 days, the differences between the two groups for all three parameters were statistically significant (P less than 0.001). The mean erythemal index decreased from 0.24 to 0.20 in the Vidida group, but increased from 0.23 to 0.25 in the Imedeen group. In the Vivida group, five patients developed transient, mild pimples during the first weeks of treatment, but no other adverse effects occurred.


Zinc gluconate and the common cold: a controlled clinical study.

A report in 1984 on the success of zinc gluconate against common cold symptoms could not be confirmed in three subsequent studies, which are now known to have used formulations that inactivated zinc. A non-chelating formulation including glycine, which releases 93% of contained zinc into saliva, was tested in a randomized, placebo-controlled, double-blind trial in 73 young adults. Efficacy was recorded in symptom diaries using a symptom severity rating. Patients' symptoms first appeared 1.34 days prior to entry to the study in both groups. Disappearance of symptoms occurred after an additional 4.9 days for zinc-treated patients versus 6.1 days for placebo-treated patients. A difference was noted in the efficacy of treatment if it was started 1 day after symptom onset: cold duration was an additional 4.3 days in zinc-treated patients compared with 9.2 days for placebo-treated patients. Cough, nasal drainage and congestion were the symptoms most affected, and only mild side-effects were noted.


Typhoid Vi: a less reactogenic vaccine.

A typhoid vaccine derived from the purified Vi capsular polysaccharide (CPS) antigen of Salmonella typhi was compared with a heat-killed whole-cell typhoid vaccine in 637 healthy male volunteers. The individuals were placed in three groups: group 1 received two doses of heat-killed whole-cell typhoid vaccine, at an interval of 28 days; group 2 received a single dose of typhoid Vi CPS vaccine followed after 28 days by water for injection; and group 3 received water for injection on the first occasion and a single dose of typhoid Vi CPS vaccine 28 days later. Local and systemic adverse reactions were recorded for 5 days following each injection. Subjects receiving the typhoid Vi CPS vaccine complained of fewer local adverse reactions on each of the first 3 days following immunization: on day 1, 18.6% of subjects given typhoid Vi CPS vaccine reported local reactions compared with 59.7% of those receiving heat-killed whole-cell vaccine (P less than 0.001). The percentage of subjects receiving the heat-killed whole-cell vaccine who complained of systemic reactions was more than twice that of subjects receiving the Vi CPS vaccine (7.9% and 3.4%, respectively, on day 1; P less than 0.01).


Cytogenetic analysis of bone marrow from patients with primary myelodysplastic syndrome.

Of 34 patients with primary myelodysplastic syndrome (MDS), 26 (76.5%) were found to have chromosome defects using an improved bone marrow culture method and high-resolution G-banding technique induced by actinomycin D. The frequency of abnormalities varied among the subtypes: 1/2 in refractory anaemia with ringed sideroblasts; 18/24 in refractory anaemia; 5/6 in refractory anaemia with excess of blasts (RAEB); and 2/2 in refractory anaemia with excess of blasts in transformation. The most frequent abnormalities, either single, double or complex defects, were -5/5q-, -7, +8 and +21; 16q-/16p-, +19 and -X were also common. The percentage of aneuploid cells, in particular hypodiploid cells, was increased. The abnormalities were detected more frequently in complex aberrations associated with RAEB and RAEB in transformation. The presence of -5/5q- as a sole aberration was associated with longer mean survival time (greater than 18 months) but multiple (more than two) chromosomal abnormalities were associated with a poorer prognosis and a mean survival time of only 7.5 months. Chromosome follow-up studies indicated that patients with -7, +8, +21, -X and complex defects, increased hypodiploid cells and karyotpic evolution were likely to have a high risk of transformation to leukaemia or to a more severe subtype of MDS with a short overall mean survival time. These defects, mostly of the deleted type, are assumed to play a specific role in the pathogenesis of myelodysplasia. Repeated chromosomal analyses during the clinical course convey more accurate prognostic criteria for patients.


Effects of pimobendan (UDCG 115) on renal function in healthy volunteers.

A phase I, double-blind, single-dose, randomized, two-period crossover study was conducted to investigate the effects of pimobendan on renal function to assess whether renal events were a contra-indication to its administration. Results in eight healthy volunteers indicated no significant adverse events on renal plasma flow, glomerular filtration rate, or laboratory safety screens; side-effects were also found to be minimal. Further studies are indicated to assess whether, in the proposed treatment group, i.e. patients with heart failure (in whom compromised renal function is common), pimobendan similarly elicits no serious adverse renal effects.


An oral approach to the treatment of photodamaged skin: a pilot study.

In recent publications much attention has focused on skin changes as a consequence of ageing. During life many internal and external factors have an influence on the condition of the skin but ultraviolet radiation seems to be a major factor in both benign and malignant alterations. To retard the deterioration process, more than cosmetic concern is necessary. In a pilot study, a fish protein product is shown to have the capacity to ameliorate, both objectively and subjectively, the symptoms of photo-aged skin. Recommendations are made for further evaluation.


Results of long-term follow-up after compensated fixed-dose therapy for thyrotoxicosis.

To evaluate the effects of simple compensated fixed-dose iodine-131 therapy for thyrotoxicosis, the long-term results for 74 patients treated with a fixed dose of iodine-131 ranging from 5 to 12 mCi (185 to 444 MBq) were evaluated in the first 2 years of a trial. The dose selected was loosely based on the gross size of the thyroid gland. Routine antithyroid drug therapy was given for a minimum of 3 months after iodine-131 therapy. The mean (+/- SD) duration of follow-up was 74.5 +/- 42 months. The results indicated that roughly 25% of patients treated in this way will become hypothyroid after 5 years and that 85% are cured (need no further therapy during the follow-up period) using a single dose of iodine-131. Of those cured using a single iodine-131 dose, 81% were no longer receiving drugs after 6 months and 85% after 1 year. Such a regimen seems currently to be among the best available where prolonged periods of medication-free euthyroidism after therapy are sought.


Platelet angiotensin II in cerebrovascular accident and the effect of angiotensin-converting enzyme inhibitors.

Platelet angiotensin II concentrations were significantly (P less than 0.01) elevated in 16 patients with a history of cerebral infarction, compared with 12 control subjects. The angiotensin-converting enzyme inhibitors, captopril, enalapril and delapril hydrochloride, were evaluated in 20 hypertensive patients with a history of cerebral infarction. Each agent was administered orally each day for 12 weeks using an open randomized crossover design. After 4 weeks' treatment, 75 mg/kg captopril significantly (P less than 0.01) increased platelet angiotensin II concentrations and the increase was maintained for a further 8 weeks. Treatment with 5 mg/day enalapril resulted in no significant change in angiotensin II. Treatment with 30 mg/day delapril hydrochloride significantly (P less than 0.05) decreased platelet angiotensin II concentrations at 4 weeks and the change persisted for 12 weeks (P less than 0.01). During delapril hydrochloride treatment platelet angiotensin II concentrations approached normal values. It is concluded that delapril hydrochloride may be used to treat patients with arteriosclerosis disorders and may inhibit the tendency for atherosclerotic changes and thrombosis.


Combined zidovudine and interferon-alpha 2a therapy in children with acquired immune deficiency syndrome.

A study was carried out in five children with acquired immune deficiency syndrome to assess the effect of combined zidovudine/interferon-alpha 2a therapy with that of zidovudine given alone on immunological profiles and plasma zidovudine concentrations. Immunoglobulins A, G and M, total and absolute CD4 lymphocyte counts, and p24 antigen concentrations did not differ significantly when children were treated with 300 mg/m2 zidovudine given orally once every 12 h, or with 150 mg/m2 zidovudine plus 1.5 or 3 MIU interferon-alpha 2a given intramuscularly three times weekly. Peak plasma zidovudine concentrations were significantly (P less than 0.05) lower when combined treatment with 150 mg/m2 zidovudine/1.5 MIU interferon-alpha 2a was administered compared with 300 mg/m2 zidovudine alone, or combined 150 mg/m2 zidovudine/3 MIU interferon-alpha 2a. The results suggest that combination zidovudine/interferon-alpha 2a therapy may be more efficacious than zidovudine alone and that the normal zidovudine dose may be reduced if interferon-alpha 2a is given in addition, thus reducing the side-effects associated with zidovudine.


Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin.

Expression of the regulatory contractile proteins, heavy caldesmon (h-caldesmon) and calponin was studied in human aortic smooth muscle cells (SMCs) during development and compared with the expression of alpha-SM-actin and smooth muscle-myosin heavy chain (SM-MHCs). For this study, novel monoclonal antibodies specific to SM-MHCs, h-caldesmon, and calponin were developed and characterized. Aortic SMCs from fetuses of 8-10 and 20-22 weeks of gestation express alpha-SM-actin and SM-MHCs, but neither h-caldesmon nor calponin were expressed as demonstrated by immunoblotting and immunofluorescence techniques. In the adult aortic tunica media, SMCs contain all four markers. Thus, the expression of calponin, similar to the expression of alpha-SM-actin, SM-MHCs, and h-caldesmon, is developmentally regulated in aortic SMCs. In the adult aortic subendothelial (preluminal) part of tunica intima, numerous cells containing SM-MHCs, but lacking h-caldesmon and calponin, were found. These results illustrate the similarity of SMCs from intimal thickenings and immature (fetal) SMCs. Expression of contractile proteins in the developing SMCs is coordinately regulated; however, distinct groups of proteins appear to exist whose expression is regulated differently. Actin and myosin, being major contractile proteins, also play a structural role and appear rather early in development, whereas caldesmon and calponin, being involved in regulation of contraction, can serve as markers of higher SMC differentiation steps. In contrast, h-caldesmon and calponin were already present in visceral SMCs (trachea, esophagus) of the 10-week-old fetus. These results demonstrate that the time course of maturation of visceral SMCs is different from that of vascular SMCs.


Factors necessary for restoring an evolutionary change in an anural ascidian embryo.

The ascidian Molgula oculata has a tailed (or urodele) larva, whereas Molgula occulta develops directly via a tailless (or anural) embryo. Interspecific hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm (M. occulta x M. oculata hybrids) can develop urodele larval structures, including a brain pigment cell and a short tail containing a small notochord. Development of larval features differs in individual M. occulta clutches: some eggs develop into hybrids with both a brain pigment cell and a tail, some into hybrids with either a brain pigment cell or a tail, and others into hybrids without urodele features. The expression of a 58-kDa protein (p58), which is present in eggs and embryos of urodele ascidians but lacking in those of most anural species, also varies in expression between different clutches of M. occulta eggs. Western blot and immunofluorescence studies show that p58 expression is correlated with the ability of hybrid embryos to express urodele features. For example, clutches of M. occulta eggs containing relatively high levels of p58 produce many hybrids with both a brain pigment cell and a tail. Differential expression of p58 occurs during oogenesis in M. occulta individuals: p58 is found at similar levels in previtellogenic oocytes, but in some animals it disappears during vitellogenesis, while in others it persists throughout oogenesis and is present in mature eggs. When M. occulta eggs are extracted with Triton X-100, p58 remains in the detergent-insoluble fraction, suggesting that it is associated with the cytoskeleton. In most unfertilized M. occulta eggs, p58 is uniformly distributed, but after fertilization it is localized in the uncleaved zygote and then concentrated in embryonic ectoderm, notochord, and muscle lineage cells. Despite containing high levels of p58, gynogenetic hybrid embryos, produced by fertilizing M. occulta eggs with uv-irradiated M. oculata sperm, develop into hybrids without a brain pigment cell or a tail. The results suggest that both a functional paternal genome and p58 are necessary for restoration of larval features in M. occulta x M. oculata hybrids. The cytoskeletal complex containing p58 may mediate the localization of key maternal factors in the egg or may be involved in cellular interactions during embryogenesis which are responsible for development of urodele cell fates.


Skeletal muscle satellite cells appear during late chicken embryogenesis.

The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult 12-16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.


Temporal appearance of satellite cells during myogenesis.

In this study, differences between fetal and adult myoblasts in clonal and high density culture have been used to determine when adult myoblasts can first be detected during avian development. The results indicate that avian adult myoblasts are apparent as a distinct population of myoblasts during the midfetal stage of development. Three different criteria were used to differentiate fetal and adult myoblasts and demonstrate when adult myoblasts become a major proportion of the myoblast population: (1) differences in slow myosin heavy chain 1 (MHC1) isoform expression, (2) initiation of DNA synthetic activity, and (3) average myoblast length. Fetal chicken (ED10-12) pectoralis muscle (PM) myoblasts form myotubes that express slow MHC1 after prolonged culture, while adult chicken PM myoblasts do not. Fetal avian myoblasts were active in DNA synthesis and large when first isolated, reaching peak rates of synthesis by 24 hr in culture, while adult myoblasts were inactive in DNA synthesis and small when first isolated, only reaching peak rates of DNA synthesis and size at 3 days of incubation. A dramatic decrease in the percentage of muscle colonies with fibers that expressed slow MHC1 was observed between the midfetal stage and hatching in the chicken, along with a corresponding decrease in myoblast DNA synthetic activity and average length during this same period in both the chicken and the quail. Myoblast activity and average length increased again 3-4 days posthatch and a small transient increase in the number of slow MHC1-expressing clones was also associated with the massive growth of muscle that occurs in the neonatal bird. We conclude that adult myoblasts are present as a distinct population of myoblasts at least as early as the midfetal stages of avian development.


The fertilization potential and associated membrane potential oscillations during the resumption of meiosis in the egg of the ascidian Phallusia mammillata.

The fertilization potential in Phallusia mammillata consisted of an initial rapid depolarization. This initial sperm-triggered depolarization was followed by a phase of membrane depolarization which was of either long or short duration, depending on the eggs. When of long duration, the phase of membrane depolarization was divided into two periods: the first one began with a plateau (Em = +20.2 +/- 1.1 mV; duration = 1.7 +/- 0.14 min) which was followed by a series of membrane potential oscillations (n = 3.1 +/- 0.25) lasting 2.4 +/- 0.2 min. The second period also began as a plateau (Em = approximately 0 mV; duration = 3.40 +/- 0.20 min) which was followed by a series of oscillations (n = 11.5 +/- 0.5) lasting 11.8 +/- 0.6 min, followed by a membrane repolarization. The second series of oscillations often continued rising from the resting potential value. In the eggs displaying a short duration of membrane depolarization, the second period of depolarization was shortened (lasting only 3.5 +/- 0.5 min) since it lacked the second plateau. In addition it displayed a smaller number of oscillations (n = 4.7 +/- 0.6). As a consequence of this shortening, the membrane repolarized sooner. After repolarization, the membrane displayed several potential oscillations that started from the repolarization level. Regardless of the length of the depolarized plateau phases, the total number of membrane oscillations and the time period during which they occurred were constant. Eggs displaying a long depolarization phase had 15.9 +/- 0.6 oscillations in a 19.5 +/- 0.6 min interval, while eggs having a short depolarization phase had 16.0 +/- 0.8 oscillations in a 18.1 +/- 0.3 min interval. The time period during which the potential oscillations occurred corresponded remarkably well with the time of the meiotic divisions: the formation of the first polar body was detected about 80 sec after the end of the first series of oscillations; the second polar body was extruded about 85 sec after the last membrane oscillation occurred.


Differentiation of retinal precursor cells born in vitro.

It is not known whether the differentiated fate of retinal precursor cells is determined before, during, or after terminal mitosis. Previous studies from this laboratory led to the hypothesis that retinal precursor cells remain plastic after final mitosis and will follow a photoreceptor "default pathway" unless induced to develop as neurons by intraretinal factors. This hypothesis predicts that isolated precursors undergoing terminal mitosis and differentiation in cell culture, in the absence of the retinal microenvironment, should become photoreceptors, regardless of embryonic age. To test this prediction precursor cells were dissociated from 5- to 8-day chick embryo retinas and grown as single cells in vitro. Bromodeoxyuridine (BRDU)- and [3H]thymidine-labeling techniques, coupled with serial photography of precursor development in culture, showed that at all donor ages some of the isolated cells divided one or more times and became postmitotic in vitro. Analysis of cell phenotype by phase-contrast microscopy, sequential photography, autoradiography, and immunocytochemistry showed that the majority of precursors from all donor ages differentiated as photoreceptors. These observations support a prediction derived from the "photoreceptor default" hypothesis.


Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development.

The hatching enzyme of medaka consists of two types of proteases (HCE, LCE). cDNA clones for LCE and HCE were isolated from a lambda gt11 cDNA library constructed with poly(A)+ RNA of Day 3 embryos. LCE cDNA is 936 bp long and contains an 813-bp open reading frame encoding a preproenzyme with a 20-amino-acid signal sequence, a 51-amino-acid propeptide, and a 200-amino-acid mature enzyme. For HCE, two distinct cDNAs (HCE21, HCE23) having nucleotide sequences with 92.8% similarity were obtained. These cDNAs contain open reading frames encoding preproenzymes of 279 and 270 amino acids, respectively. The mature enzyme forms of both consist of 200 amino acids, the similarity between them being 95.5%. On Northern blotting analysis, the transcripts of LCE and HCE genes were first detected coincidentally in Day 2 embryos shortly before the production of LCE and HCE, accumulated thereafter in parallel, and dramatically decreased after hatching. The amino acid sequence, the HExxH motif, which is known to constitute an active site in some Zn proteases, is also found in LCE and HCE. However, the sequence analyses strongly suggest that both the enzymes belong to the astacin (protease) family, being distinct from sea urchin hatching enzyme, which is reportedly similar to collagenase.


The repetitive calcium waves in the fertilized ascidian egg are initiated near the vegetal pole by a cortical pacemaker.

Ascidian eggs respond to fertilization with a series of repetitive calcium waves that originate mostly from the vegetal/contraction pole region (J. E. Speksnijder, C. Sardet, and L. F. Jaffe, 1990, Dev. Biol. 142, 246-249), where the myoplasm is concentrated during the first phase of ooplasmic segregation. This suggests that the myoplasm may be involved in initiating these calcium waves. To test this possibility, the starting position of the calcium waves was determined in eggs that had the subcortical, mitochondria-rich part of the myoplasm displaced by centrifugation. Such centrifuged eggs display four cytoplasmic layers: a large centrifugal yolk zone, a narrow clear zone, a mitochondria-rich layer, and a small clear zone at the centripetal pole. Imaging of the cytosolic calcium in centrifuged eggs that were injected with the calcium-specific photoprotein aequorin reveals a series of repetitive calcium waves after fertilization. About 70% of these waves start in the vegetal/contraction pole area, which is similar to the number of waves previously found to start in this area in uncentrifuged eggs. In contrast, only about 25% of the waves start close to the displaced mitochondria-rich layer. From this result it is concluded that the main wave initiation site is not displaced by the centrifugal forces that displace the subcortical, mitochondria-rich part of the myoplasm. Moreover, the observation that the animal-vegetal polarity of cortical components such as actin filaments and the endoplasmic reticulum has been retained after centrifugation further suggests that a cortical component located in the vegetal hemisphere--most likely the endoplasmic reticulum network in the cortical region of the myoplasm--is involved in initiating the repetitive calcium waves in the fertilized ascidian egg.


Changes in voltage-dependent ion currents during meiosis and first mitosis in eggs of an ascidian.

Different patterns of voltage-dependent ion currents are present in mature eggs and in early embryos of the ascidian Boltenia villosa, as if each ion current is regulated in a different manner between fertilization and the early cleavages of embryogenesis. The ion currents appear and/or disappear with precise timing suggesting that they play important roles at specific times during early development. We investigated changes in three voltage-dependent ion currents (an inwardly rectifying chloride current, a calcium current, and a sodium current) and membrane surface area over time between the resumption of meiosis (with fertilization or activation) and the first mitotic cleavage. Using time-lapse video recordings made during whole-cell patch-clamp experiments, we were able to correlate electrophysiological changes with morphological changes and cell cycle related events. Between fertilization and first cleavage, INa was lost exponentially, the density of ICa remained relatively constant, and the amplitudes of both ICl and membrane surface area fluctuated in time with the cell cycle. ICl and surface area increased whenever the cell began dividing--with the polar body extrusions and the formation of the first cleavage furrow. This suggested that the values of ICl and surface area were largest during interphase and smallest during M-phase of each cell cycle. This hypothesis was supported by an experiment in which entry into M-phase was blocked in fertilized eggs by inhibiting protein synthesis. This prevented the decreases of ICl and surface area but allowed the increases to occur normally. Patterns of change in ion currents are current specific and, as is the case with ICl, are tightly correlated with developmental events.


Maturation-specific deadenylation in Xenopus oocytes requires nuclear and cytoplasmic factors.

During the meiotic maturation of Xenopus oocytes, maternal mRNAs that lack a cytoplasmic polyadenylation element are deadenylated and translationally inactivated. In this report, we have characterized the regulation of poly(A) removal during maturation. Deadenylation in vivo is detected only after germinal vesicle breakdown and does not require de novo protein synthesis. Enucleated oocytes do not deadenylate either endogenous or microinjected RNAs upon maturation, indicating that a nuclear component is required for poly(A) removal. Whole cell extracts prepared from both immature and mature oocytes deadenylate exogenous RNA substrates in vitro. Deadenylation activity is not detected in isolated nuclear or cytoplasmic extracts obtained from immature oocytes, but is reconstituted when these fractions are combined in vitro. These results indicate that the factors required for deadenylation activity are present in immature oocytes, but that poly(A) removal is prevented by the sequestration of one or more of these components within the nucleus. Maturation-specific deadenylation of maternal mRNAs occurs upon the release of nuclear factors into the cytoplasm at germinal vesicle breakdown.


Effects of antibodies against N-cadherin and N-CAM on the cranial neural crest and neural tube.

We have examined the distribution and function of the defined cell adhesion molecules, N-cadherin and N-CAM, in the emigration of cranial neural crest cells from the neural tube in vivo. By immunocytochemical analysis, both N-cadherin and N-CAM were detected on the cranial neural folds prior to neural tube closure. After closure of the neural tube, presumptive cranial neural crest cells within the dorsal aspect of the neural tube had bright N-CAM and weak N-cadherin immunoreactivity. By the 10- to 11-somite stage, N-cadherin was prominent on all neural tube cells with the exception of the dorsal-most cells, which had little or no detectable immunoreactivity. N-CAM, but not N-cadherin, was observed on some migrating neural crest cells after their departure from the cranial neural tube. To examine the functional significance of these molecules, perturbation experiments were performed by injecting antibodies against N-CAM or N-cadherin into the cranial mesenchyme adjacent to the midbrain. Fab' fragments or whole IgGs of monoclonal and polyclonal antibodies against N-CAM caused abnormalities in the cranial neural tube and neural crest. Predominantly observed defects included neural crest cells in ectopic locations, both within and external to the neural tube, and mildly deformed neural tubes containing some dissociating cells. A monoclonal antibody against N-cadherin also disrupted cranial development, with the major defect being grossly distorted neural tubes and some ectopic neural crest cells outside of the neural tube. In contrast, nonblocking N-CAM antibodies and control IgGs had few effects. Embryos appeared to be sensitive to the N-CAM and N-cadherin antibodies for a limited developmental period from the neural fold to the 9-somite stage, with older embryos no longer displaying defects after antibody injection. These results suggest that the cell adhesion molecules N-CAM and N-cadherin are important for the normal integrity of the cranial neural tube and for the emigration of neural crest cells. Because cell-matrix interactions also are required for proper emigration of cranial neural crest cells, the results suggest that the balance between cell-cell and cell-matrix adhesion may be critical for this process.


Spatial and temporal transcellular current patterns during oogenesis.

We have used the two-dimensional vibrating probe to examine spatial and temporal patterns in the transcellular current flow around telotrophic ovarioles of the insect Rhodnius prolixus. We demonstrate a dynamic pattern of currents which correlates with various stages of vitellogenesis. Asymmetries exist in the radial current pattern around intact ovarioles, particularly around the terminal follicle, and may correlate with early developmental axes. The extra-cellular current pattern is largely reflected by a similar, though weaker pattern of currents over the germ cell membranes, indicating that both germ cell and somatic cell membranes are involved in current generation. Current enters previtellogenic oocytes and leaves oocytes entering vitellogenesis. We speculate that current reversal and loss of trophic cord contact may represent an electrophysiological feedback control mechanism during oogenesis.


Exogenous transferrin is taken up and localized by the neurulation-stage mouse embryo in vitro.

We have screened neurulation-stage mouse embryos for regional differences in protein distribution, by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The screen has revealed an 83-kD protein (pI 6.8) that is present in embryo regions where neurulation is in progress but not in regions where neurulation is complete. The 83-kD protein is not synthesized in the neurulation-stage embryo or in the yolk sac, but is taken up from the culture serum in vitro and, probably, from the maternal serum in utero. The 83-kD protein has been identified as transferrin on the basis of its electrophoretic migration and recognition on Western blots by an antitransferrin antibody. Culture of embryos in serum containing 125I-transferrin, followed by autoradiography of embryo sections, shows that transferrin is taken up and localized in the gut beneath the closing neural folds at several levels of the body axis in 8.5- and 9.5-day embryos. In situ hybridization studies show that the transferrin receptor mRNA is expressed in all cells of the 9.5-day embryo, including the gut endoderm. These findings are consistent with a role for transferrin in development of the gut and perhaps, indirectly, in completion of neurulation during early mouse embryogenesis.