Is your ribozyme design really correct?: A proposal of simple single turnover competition assay to evaluate ribozymes.

Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.

Factor analysis of the components of 12 standard test batteries, for unilateral spatial neglect, reveals that they contain a number of discrete and important clinical variables.

The aim of this study was to investigate a conventional battery of tests capable of assessing the presence of the component and extent of the lesions in patients with unilateral spatial neglect. Ninety-four patients who had unilateral spatial neglect with a stroke in right hemisphere were assessed on 12 traditional neglect batteries 4 weeks after the onset. Computerized tomography was also performed to investigate the possible anatomical relationships with each neglect battery. Factor analysis showed that the tests loaded significantly on five factors. There are not only visual scanning factors but also factors of imaging, visual judgement, visual cognition and effectiveness from left hemisphere in the unilateral spatial neglect. There are high correlations between each neuropsychological test and neglect batteries. Furthermore, lesions in the paraventricular white matter were associated with clock and person drawing tasks. Lesions in the occipital lobe were associated with reading, explaining and visual counting tasks. Lesions in the temporal lobe and the posterior limb of the internal capsule were associated with line bisection tasks. It is suggested that it is possible that there are some different components in unilateral spatial neglect. Failure in some tasks may predict different lesions in terms which include localization.

Apraxia and cerebral haemorrhage: the relationship between haematoma volume and prognosis.

In this study, we examined twenty-five patients with left putaminal haemorrhage to investigate the relations between ideational apraxia (IA) and intracerebral haemorrhage. Apraxias were determined at 1 and 6 months after the stroke onset. Extension and volume of haematoma were examined with CT scan within 2 days of stroke onset. IA was present in 10 cases at 1 month and disappeared in 6 cases (transient IA) and persisted in 4 cases(persisted IA) at 6 months from the onset. Although the haematoma volume related to the existence of IA, there was no significant difference between transient and persistent IA. All three patients with the haematoma volume larger than 40 ml in the transient IA group were younger than 50 years old. All cases with persistent IA were older than 50 years old. Consequently, the existence of IA seems to be partially dependent on the haematoma volume which may cause the organic damage of subcortical white matter. However, patient's age is also important to determine the prognosis of IA. This maybe related to the nature of the haemorrhage and the mode of the onset. These factors remain to be determined.

Comparative study of two ribozymes and DNA-enzyme against the same RNA target.

Two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme were designed to cleave a same RNA target, the same site of the rat complement regulatory factor 512 antigen mRNA. The kinetic properties of these RNA-cleaving enzymes were measured and compared under the same conditions, using multiple turnover kinetics and competition kinetics. The catalytic efficiencies of these enzymes, and also the order of these enzymes will be discussed.

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs.

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.

Sheath proteins: synthesis, secretion, degradation and fate in forming enamel.

We investigated expression of ameloblastin and sheathlin, recently cloned enamel matrix proteins from the rat and pig, in forming enamel immunocytochemically and immunochemically, using region-specific antibodies. The results obtained from the rat and pig were essentially the same. Antibodies which recognize the N-terminal region stained the secretory machinery of the secretory ameloblast and the entire thickness of the enamel matrix, especially the peripheral region of the enamel rod. Immunostained protein bands were observed near 65 or 70 kDa and below 20 kDa. C-terminal-specific antibodies stained the secretory machinery of the ameloblast and the immature enamel adjacent to the secretion sites. Immunostained protein bands were found ranging from 25 to 70 kDa. Antibodies which recognize a region in the protein just prior to the C-terminal region stained the cis-side of the Golgi apparatus but not the enamel matrix. Immunostained protein bands were observed of about 55 kDa. These results suggest that post-translational and post-secretory modifications of ameloblastin and sheathlin are similar to each other, and further showed that their cleaved N-terminal polypeptides concentrate in the prism sheath. We propose that sheathlin and ameloblastin share the same role in amelogenesis and should be classified as sheath proteins.

Effect of dietary linoleate/alpha-linolenate balance on experimentally induced gastric injury in rats.

Rats were fed diets containing different ratios of linoleate (18:2 n-6) to alpha-linolenate (18:3 n-3), and the severity of gastric injury induced by ethanol, ischemia/reperfusion and water-immersion stress was compared. On decreasing the 18:2 n-6/18:3 n-3 ratios in the diets, the proportion of arachidonic acid (20:4 n-6) decreased and that of eicosapentaenoic acid (20:5 n-3) increased in the phospholipids of the gastric mucosa, which was associated with decreased mucosal prostaglandin E2 content. Mucosal injury in all the three experimental models was exacerbated significantly in the diet group fed 18:2 n-6/18:3 n-3 ratio of 0.25 (perilla oil) as compared with the groups fed dietary oils with 18:2 n-6/18:3 n-3 ratios of 2.7 (mixture of perilla and safflower oils) and 127 (safflower oil). This adverse effect induced by perilla oil diet was not observed when rats were pretreated with a mild irritant (20% ethanol) prior to challenge with a strong irritant (absolute ethanol). Furthermore, an 18:2 n-6/18:3 n-3 ratio of as low as 1 was found to be in a safe range in the water-immersion stress ulcer model. Thus, oils with very low n-6/n-3 ratios, for example perilla oil, could be used without the risk of the observed adverse effects on experimental gastric injury in people of industrialized countries ingesting foods with n-6/n-3 ratios of above 4. A decrease in the n-6/n-3 ratios to 2 or below is still recommended for the prevention of chronic diseases in the elderly related to atherosclerosis and inflammation.

Cloning and characterization of porcine enamelin mRNAs.

Dental enamel forms by matrix-mediated biomineralization. The components of the developing enamel matrix are generally specific for that matrix. The primary structures of three enamel proteins-amelogenin, tuftelin, and sheathlin (ameloblastin/amelin)-have been derived from cDNA sequences. Here we report the cloning and characterization of mRNA encoding a fourth enamel protein: enamelin. The longest porcine enamelin cDNA clone has 3907 nucleotides, exclusive of the poly(A) tail. The primary structure of the secreted protein is 1104 amino acids in length. Without post-translational modifications, the secreted protein has an isotope-averaged molecular mass of 124.3 kDa and an isoelectric point of 6.5. Polymerase chain-reaction phenotyping of enamelin cDNA suggests that porcine enamelin transcripts are not alternatively spliced and use a single polyadenylation/cleavage site. Immunohistochemical and Western blot analyses with an affinity-purified antipeptide antibody specific for the enamelin carboxyl terminus demonstrate that enamelin is synthesized and secreted by secretory-phase ameloblasts. The parent protein is a 186-kDa glycoprotein that concentrates along the secretory face of the ameloblast Tomes' process. Intact enamelin and proteolytic cleavage products containing its carboxyl terminus are limited to the most superficial layer of the developing enamel matrix, while other enamelin cleavage products are observed in deeper enamel.

Buccofacial apraxia and left cerebral haemorrhage.

We examined 33 patients with left intracerebral haemorrhage to investigate the relations to buccofacial apraxia (BFA). BFA was present in 18 cases at 1 month and disappeared in 3 cases and persisted in 15 cases at 6 month from the onset. The existence of BFA seems to be partially dependent the haematoma volume which may cause the organic damage in lenticulate nucleus, insula, posterior limb or internal capsule and anterior portion of paraventricle white matter. All patients with BFA had aphasia (Broca 6, Wernicke 1, Total 10, Amnestic 1). Aphasia of the patients with transient BFA improved as well as the patients without BFA after BFA disappeared. We suspected that improvement or aphasia might be affected by BFA.

Synthesis, secretion, degradation, and fate of ameloblastin during the matrix formation stage of the rat incisor as shown by immunocytochemistry and immunochemistry using region-specific antibodies.

Rat ameloblastin is a recently cloned tooth-specific enamel matrix protein containing 422 amino acid residues. We investigated the expression of this protein during the matrix formation stage of the rat incisor immunohistochemically and immunochemically, using anti-synthetic peptide antibodies that recognize residues 27-47 (Nt), 98-107 (M-1), 224-232 (M-2), 386-399 (M-3), and 406-419 (Ct) of ameloblastin. Immunohistochemical preparations using antibodies Nt and M-1 stained the Golgi apparatus and secretory granules of the secretory ameloblast and the entire thickness of the enamel matrix. Only M-1 intensely stained the peripheral region of the enamel rods. Immunostained protein bands were observed near 65, 55, and below 22 kD. Immunohistochemical preparations using antibodies M-2 and Ct stained the Golgi apparatus and secretory granules of the ameloblast and the immature enamel adjacent to the secretion sites, but not deeper enamel layers. Immunostaining using M-2 and Ct revealed protein bands near 65 and 40-56 kD, and 65, 55, 48, 36, and 25 kD, respectively. M-3 stained the cis side of the Golgi apparatus but not the enamel matrix. This antibody recognized a protein band near 55 kD, but none larger. After brefeldin A treatment, immunoreaction of the 55-kD protein band intensified, and dilated cisternae of rER of the secretory ameloblast contained immunoreactive material irrespective of the antibodies used. These data indicate that ameloblastin is synthesized as a 55-kD core protein and then is post-translationally modified with O-linked oligosaccharides to become the 65-kD secretory form. Initial cleavages of the 65-kD protein generate N-terminal polypeptides, some of which concentrate in the prism sheath, and C-terminal polypeptides, which are rapidly degraded and lost from the enamel matrix soon after secretion.

Rehabilitation of patients with anosognosia for hemiplegia due to intracerebral haemorrhage.

We have studied the differences in the lesions, concomitant symptoms and ADL levels in patients in acute-stage cerebral-haemorrhage and manifesting anosognosia. Twelve of 50 patients (24.0%) presented with anosognosia. The (+) group had longer intervals between onset and the first evaluation than did the (-) group, demonstrating severe sensory disturbance. The volume of haemorrhage was significantly larger in the (+) group. Anosognosia disappeared within 3 months in all cases. In the (+) group the time until discharge was long, with the ADL level at the time of discharge being low. Therefore, it was considered that anosognosia was important as an inhibitory factor hampering rehabilitation.

Is unilateral spatial neglect a single phenomenon? A comparative study between exploratory-motor and visual-counting tests.

The aim of this study is to report the preliminary findings of a traditional battery of tests and our original battery capable of assessing the presence of components and extent of lesions in patients with unilateral spatial neglect. Thirty patients who had unilateral spatial neglect with a stroke in the right hemisphere were assessed for unilateral spatial neglect on exploratory-motor (E-M) tasks, visual-counting (V-C) tasks, and traditional neglect batteries at least 4 weeks after the onset. Other neuropsychological tests and computed tomography were also performed to investigate the relationship with neglect. A factor analysis showed that our tasks loaded significantly on three factors. E-M neglect was found in 16 patients, and V-C neglect in 22 patients with unilateral spatial neglect. There were high correlations between E-M neglect and motor paralysis, word fluency, backward digit span and motor impersistence. There were high correlations between V-C neglect and visual-field defect, line bisection, line cancellation and figure copying. Lesions in the frontal lobe, caudate, insula, and anterior portion of the paraventricular white matter were commonly associated with E-M neglect. Lesions in the occipital lobe were also associated with V-C neglect. We suggest that unilateral neglect is not a single phenomenon, but rather involves several different components. We propose that E-M and V-C tasks are useful methods for evaluating the extent of lesions in patients with unilateral spatial neglect.

Immunochemical and immunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related proteins in the porcine tooth germ.

Our previous report identified 27- and 29-kDa calcium-binding proteins in porcine immature dental enamel. In this study we revealed that the N-terminal amino acid sequences of the two proteins were identical: LLANPXGXIPNLARGPAGRSRGPPG. The sequence matches a portion of the amino acid sequence of the porcine sheath protein, sheathlin. Porcine tooth germs were investigated immunochemically and immunohistochemically using specific antibodies raised against synthetic peptide that included residues 13-25 of this sequence. The affinity-purified antibodies reacted with several proteins extracted from newly formed immature enamel in immunochemical analyses, especially protein bands migrating at 62, 35-45, 29, and 27 kDa in SDS-polyacrylamide gels. The largest protein detected was a weak band near 70 kDa. In immunochemical analyses of proteins extracted from the inner (old) immature enamel, the antibody reacted faintly with the 27- and 29-kDa proteins. In immunohistochemical preparations, the Golgi apparatus and secretory granules of the secretory ameloblast, and the surface layer of immature enamel showed immunoreactivity. The immunoreactivity of immature enamel just beneath the secretory face of the Tomes' process was intense. No immunoreactivity was found in the Golgi apparatus of the maturation ameloblast. These results suggest that the 70-kDa protein, whose degradation might be very fast, is the parent protein of the 27- and 29-kDa proteins.

Exploratory-motor task to evaluate right frontal lobe damage.

An exploratory-motor (E-M) task was performed by 20 patients with right hemispheric damage and unilateral spatial neglect and by 15 adult controls. None of the subjects in the control group demonstrated E-M neglect, other types of unilateral spatial neglect, or motor impersistence. In the unilateral spatial neglect group, 14 of 20 patients revealed E-M neglect. The presence or absence of E-M neglect related to word fluency, digit span recall (backward), and motor impersistence. In addition, E-M neglect frequency was observed in patients with lesions in the frontal lobe. The frontal lobe may play an important role in the performance of an E-M task. Therefore, we think that E-M task performance is a useful method for evaluating the site of right frontal lobe damage.

Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins.

Sheath proteins designate low-molecular-weight non-amelogenin enamel polypeptides and their parent protein, which concentrate in the sheath space separating rod and inter-rod enamel (Uchida et al., 1995). Two porcine sheath proteins, with apparent molecular weights of 13 and 15 kDa, are characterized by protein sequencing. The primary structures of these polypeptides match a portion of the derived amino acid sequences of clones isolated from a porcine enamel organ epithelia-specific cDNA library. Sheath protein RNA messages differ by the inclusion or deletion of a 45-nucleotide segment and by the use of three alternative polyadenylation/cleavage sites. The secreted proteins are 395 and 380 residues in length, with molecular masses of 42,358 and 40,279 Daltons and calculated isoelectric points of 6.3 and 6.7, respectively. Polyclonal antibodies were raised against a synthetic peptide having the sheathlin-specific sequence EHETQQYEYSGGC. Immunohistochemistry with this antibody demonstrates that the protein encoded by the sheathlin cDNA is preferentially localized in the sheath space. We propose that the porcine sheath proteins and their proteolytic cleavage products be designated "sheathlin".

Design and in vitro activity of ribozymes against mRNA of a rat membrane inhibitor of complement.

Mammalian cells are usually protected from the complement-mediated injury by a number of complement regulatory proteins. A rat protein designated as 512 antigen is thought to be such a complement regulatory protein. A cDNA of 512 antigen has been cloned and analyzed. To investigate the function of 512 antigen, we plan to construct a ribozyme system against 512 antigen expression. We have designed two hammerhead ribozymes targeted to the 512 antigen mRNA and tested the ribozyme activities in vitro. Both hammerhead ribozymes efficiently cleaved the 512 antigen mRNA in vitro. We have also designed a hairpin ribozyme against this mRNA. The activity of the hairpin ribozyme will also be discussed.

Functional outcome following thalamic haemorrhage: relationship between motor and cognitive functions and ADL.

Twenty-two patients with thalamic haemorrhage were examined to investigate the relationship between motor and cognitive function, and activities of daily living (ADL). Patients with unilateral spatial neglect had lower ADL scores on admission than patients without unilateral spatial neglect (Mean: 17.0 and 24.6, respectively; F = 4.38, df = 1, p < 0.05). Unilateral spatial neglect related to feeding, bowel control and transfer in Barthel index on admission. Patients with aphasia on admission had lower ADL at discharge than patients without aphasia on admission (Mean: 57.0 and 84.7, respectively; F = 7.70, df = 1, p < 0.05). Aphasia related to the bathing, toilet, stair climbing, dressing, and ambulation in Barthel index on discharge. There was a significant difference between the severity of paresis in upper and lower limb on admission and ADL at discharge. The two-way repeated measures ANOVA showed a significant difference between severity of paresis in lower limb and ADL improvement. It can be suggested that the most important predictor of outcome was paresis in lower limb, and not aphasia or unilateral spatial neglect.

Primary structure of the porcine 89-kDa enamelin.

The primary structure of the 89-kDa enamelin found in porcine secretory enamel at an early stage of development was investigated. The fragments of the enamelin cDNA were amplified by polymerase chain-reaction from the first-strand enamelin cDNA, and were sequenced. The results indicated that the 89-kDa enamelin consisted of 627 amino acid residues and had a molecular mass of 70,448. A hydrophobic domain is located in the region of the 21st-62nd amino acid residues of the molecule. Acidic domains are located in two regions of the molecule-one in the region of the 135th-238th amino acid residues and the other in the C-terminal region. A basic domain is located in the region of the 239th-360th amino acid residues. The results also indicated that the low-molecular-weight enamelins were fragments derived from a prototype enamelin.

Transcriptional and post-transcriptional regulation of pr22 (Op18) with proliferation control.

The pr22 gene was isolated as a gene which is expressed in proliferating cells but not in cells which are differentiated or growth-arrested. When cells of the human monocytic cell line, U937, were differentiated into macrophages, transcription of pr22 was almost completely suppressed. Serum starvation resulted in the inhibition of transcription, although U937 failed to differentiate. In a culture synchronized with excess thymidine, mRNA of pr22 was detected at the G1/S boundary, with the level increasing in the S phase and decreasing in the G2 phase. The gene product, pr22 protein (Pr22) was found to be identical to Op18 as well as to a catastrophe factor. Genes homologous to pr22 were detected in the genome of mouse but not in that of yeast, or Drosophila. The 5' up-stream region of the genomic pr22 contained CpG islands but no TATA box at its appropriate position. About 20% of cell nuclei of normal human fibroblasts were stained in a speckled manner with a monoclonal antibody for C-terminal peptide of Pr22, and these cells were found to be in phases S and G2. The mitotic apparatus was also strongly stained. By Western blot analysis, Pr22 was detected in the nuclear fraction but not in the cytoplasm. The level increased from middle S to G2 phase and remained high until the early G1 phase. N-terminal truncated Pr22 was also detected in these phases. These results suggest that Pr22 may have an additional role other than just functioning in association with microtubules.

Molecular cloning of the rat complement regulatory protein, 5I2 antigen.

We have cloned a cDNA which encoded for rat complement regulatory protein, 5I2 antigen. The deduced amino acid sequence of 497 residues predicted essentially the same domain structure as the mouse complement regulatory protein, Crry/p65, composed of five short consensus repeat (SCR) domains, a transmembrane domain and a cytoplasmic domain. 5I2 antigen, however, had an additional SCR which showed a close similarity to the sixth SCR of mouse complement receptor 1 (CR1) or to the sixth, thirteenth, twentieth and twenty seventh SCR of human CR1. Corresponding domains of 5I2 antigen and mouse Crry/p65 showed 70%-86% amino acid identity, indicating that 5I2 antigen is the rat counterpart of mouse Crry/p65.