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The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.

To determine whether the nocturnal decrease in arginine vasopressin (AVP) secretion into the plasma, found in a patient with multiple system atrophy (MSA) reported previously, is a usual finding in MSA, the plasma AVP concentrations in 13 patients with MSA were measured every 4 hours during a 24 hour period. The plasma AVP concentrations in these patients showed significant daily variations and were the lowest during the night. This finding indicates that patients with MSA often exhibit nocturnal decrease in AVP secretion into the plasma. The results suggest the possibility that the system responsible for the daily variations in AVP secretion is involved in MSA.

Histological, ultrastructural, and morphometric studies were performed on nerve and muscle biopsies from three patients with de Sanctis-Cacchione syndrome. Sural nerves showed marked loss of the myelinated fibers, in proportion to decrease in nerve conduction velocities and in inverse proportion to the severity of the clinical symptoms, which were related to the survival length. The larger fibers were involved earlier and more markedly than the smaller. The unmyelinated fibers were also decreased in number. Electron-microscopic studies showed the presence of primary degeneration of myelin sheaths or Schwann cells. Muscle biopsies showed grouping of type I and type II fibers in all three patients. Therefore, peripheral nerve involvement in de Sanctis-Cacchione syndrome was suggested to result from chronic degeneration of the neuronal cells and Schwann cells.

The structural polypeptides of the murine coronavirus DVIM (diarrhoea virus of infant mice) have been analysed in comparison with other strains MHV-2, MHV-3, MHV-4 (JHM) and MHV-S by SDS-PAGE. In the presence of 2-mercaptoethanol, three major glycopolypeptides, gp180, gp69, gp25 (as a group of similar species) and one major non-glycosylated polypeptide p58 were detected. The gp69 is a DVIM specific glycopolypeptide, in which the glycosidic moieties are linked to the core polypeptide through N-glycosidic bonds, and hence may be correlated with the short projections of the viral envelope. Further gp140, which appears in the absence of reducing agents, is apparently a dimer of gp69 held together by disulfide linkages. The gp25 family, on the other hand, consists of four polypeptides, two of which are not metabolically inhibited by tunicamycin suggesting that they are O-linked glycopolypeptides. DVIM seems to be serologically closely related to the MHV-S strain as shown by neutralization.

Summary The effects of five lysosomotropic drugs (NH4Cl, chloroquine, methylamine, amantadine and dansylcadaverine) and cytochalasin B on human rotavirus (HRV KUN strain) and vesicular stomatitis virus (VSV Indiana strain) infection in monkey MA 104 cells were examined. These drugs had little effect on HRV yield but greatly reduced VSV yield. The results strongly suggest that HRV does not require endocytotic activity and intracellular acidic vesicles for the initial stage of infection and support our postulate that HRV enters the target cell by direct penetration of its nucleoid through the cell membrane.

Ultrastructural studies on muscle biopsies from three patients with Becker's muscular dystrophy showed that the i.m. nerves presented loss or disarrangement of the neurofilaments and an increased number of glycogen granules and/or myelin figures not infrequently in the myelinated and unmyelinated nerve fibers. The neuromuscular junctions showed markedly widened sole-plate areas, and several terminal axons frequently abutted and formed neuromuscular junctions on the same fiber. The secondary synaptic clefts were markedly decreased in number and short in length in type I fibers but not in type II fibers. Most terminal axons showed no degenerative changes. Therefore, the participation of a neural factor might be suggested as the cause of Becker's muscular dystrophy, although it does not mean denervation in the conventional sense of an axonal degeneration.

Picornavirus-like crystalline arrays were found in the skeletal muscle of a victim of a traffic accident. Individual particles measured approximately 22 nm in diameter and were arrayed in a hexagonal or triangular pattern. Cis-platinum (II) staining of these virus-like particles showed that these particles were not glycogen but contained nucleic acids, and the staining according to Bernhard also showed these particles contained RNA. Through the combination of the methods of Heinen (1977) and Bernahrd (1969), we can confirm that virus-like crystalline structures found electron microscopically contain nucleic acids and possibly represent virions.

We used histologic evidence of degenerative changes in both the gray and white matter of the brain to diagnose a patient as having the panencephalopathic type of Creutzfeldt-Jakob disease (CJD). This type of CJD is relatively common in Japan, but not in North America or Europe. We recovered a transmissible pathogen (Echigo-1 strain) from an autopsy specimen of the patient's brain and passed it serially in Hartley guinea pigs. After a long latent period, it caused degenerative changes, mainly in the thalamic area of the guinea pig brain. On the 4th passage, a substrain emerged with a short latent period. When cross-transmitted to Golden Syrian hamsters, this substrain induced severe degeneration in both the thalamus and cerebral cortex. We compare our results with those for other experimental CJDs produced by other types of this disease.

TAL1, which is frequently activated in T cell acute lymphoblastic leukemia (T-ALL), encodes lineage-specific basic helix-loop-helix (bHLH) proteins that bind specifically to E-box DNA motif upon dimerization with ubiquitous basic helix-loop-helix proteins E47 or E12. RBTN1 and RBTN2, also frequently activated in T-ALL, encode proteins only with tandem cysteine-rich LIM domains. We found that aberrant expression of TAL1 detected in 11 out of 14 T-ALL cell lines was invariably accompanied by that of either RBTN1 or RBTN2. Forced expression of TAL1 together with RBTN1 or RBTN2, but not TAL1 alone, strongly induced artificial reporter genes in a TAL1/RBTN-negative T-ALL cell line, HPB-ALL. Such collaborative transcriptional activity of TAL1 and RBTN was not, however, observed in non-T cell lines, suggesting further involvement of some T cell-specific cofactors. In this context, we carried out preliminary evaluation of a potential role of the T cell-specific GATA-binding protein, GATA3, in the transcriptional activity of TAL1 and RBTN. We also showed that coexpression of TAL1 and RBTN1 in HPB-ALL strongly induced TALLA1, a highly specific T-ALL marker whose positivity correlated 100% with ectopic expression of TAL1 among various T-ALL cell lines. Collectively, ectopic TAL1 and RBTN1 or -2, together with some endogenous T cell-specific cofactors like GATA3, constitute a highly collaborative set of transcription factors whose aberrant activity in T cells may lead to leukemogenesis by modulating expression of downstream genes such as TALLA1.

A cell lysate prepared from MA104 cells that had been infected with human rotavirus KUN strain (HRV-KUN) contained a 35-kilodalton protein capable of binding to MA104 cells. The binding of the 35-kilodalton protein was inhibited by a serotype 2-specific antiserum but not by antisera to other serotypes. Not only trypsin-treated, infectious HRV-KUN but also untreated, noninfectious virions effectively competed with the 35-kilodalton protein for the same cell surface binding sites. One monoclonal anti-VP7 (AH6) absorbed the 35-kilodalton protein from the HRV-KUN-infected cell lysate, whereas another monoclonal anti-VP7 (S2-2G10) inhibited the virions to compete with the 35-kilodalton protein for the cell surface binding sites. Both anti-VP7 (S2-2G10) and anti-VP3 (K-1532, K-376) monoclonal antibodies had the virus-neutralization activity, but only anti-VP7 inhibited virus adsorption. On the other hand, anti-VP3 monoclonal antibodies were capable of completely inhibiting the infection of preadsorbed HRV-KUN as long as virions were not yet internalized. Subsequent studies with [35S]methionine-labeled and purified HRV-KUN showed that not only trypsin-treated, infectious virions but also untreated, noninfectious virions were capable of efficient target cell binding and internalization. The internalization modes of these two HRV-KUN preparations were, however, quite different. Only the components of the inner capsid were internalized from trypsin-treated virions, whereas no such selective internalization was seen with untreated virions. Furthermore, anti-VP3 inhibited this selective internalization of the inner capsid from the infectious virions. From these results we conclude that VP7 is the HRV-KUN cell attachment protein and that adsorption of HRV-KUN via VP7 is independent of trypsin treatment, whereas the limited cleavage of VP3 by trypsin, which is essential for the development of HRV-KUN infectivity, is needed for the selective internalization of the inner capsid components, a process that is apparently essential for HRV-KUN infection.

Daily injection of methamphetamine (MAP) increased the locomotor activity and produced the development of MAP-induced anticipatory activity on the next withdrawal day. Daily restricted feeding also causes a feeding-associated anticipatory activity rhythm. The importance of catecholaminergic neurons has been suggested in the manifestation of a feeding-associated corticosterone rhythm. In our study, we examined the role of dopaminergic neurons in the development of MAP-induced anticipatory activity. The development of MAP-induced anticipatory activity was blocked by coadministration of dopamine D2 receptor antagonists such as YM-09151-2 and sulpiride, the D1 receptor antagonist SCH23390 and haloperidol weak D1/D2 receptor antagonist, but not by clozapine. The anticipatory activity increase was reproduced by daily injection of the D2 receptor agonist quinpirole as well as moderately by the D1 receptor agonist SKF38393. Moreover, MAP-induced anticipation was blocked by coadministration of the N-methyl-D-aspartate receptor antagonist MK-801 or sigma ligands rimcazol and NE-100. Our results suggest that activation of the dopaminergic system, especially via D2 receptors may be required for the development of MAP-associated anticipatory activity increases and that N-methyl-D-aspartate receptors and sigma receptor mechanisms are also involved in the development of this behavior.

The authors describe a patient who had a point mutation at codon 232 of the prion protein gene, resulting in the substitution of methionine for arginine (M232R). The patient developed dementia and died 6 years after its onset. Autopsy revealed dementia with Lewy bodies, not Creutzfeldt–Jakob disease. Although the M232R mutation has been reported to cause Creutzfeldt–Jakob disease, findings in our patient suggest that not all patients presenting progressive dementia with M232R mutation have Creutzfeldt–Jakob disease.

We documented the presence of a point mutation in the tRNALysgene of mitochondrial DNA (mtDNA) in various postmortem tissues from two patients with myoclonus epilepsy associated with ragged-red fibers (MERRF). The percentages of the mutant mtDNA were similar (93 to 99) in both clinically affected and unaffected tissues, suggesting that preferential clinical involvement of certain tissues in MERRF is based not only on the variation of distribution of the mutant mtDNA, but also on other factors such as differences in the threshold in various CNS regions and organs.

A case of adrenoleukodystrophy showing neurological features of olivopontocerebellar atrophy is described. A CT scan demonstrated marked atrophy in both cerebellum and pons. ACTH stimulation produced no rise in the plasma cortisol level but a significant rise in the plasma aldosterone level. The ratios of C26:0 to C22:0 in fatty acids of sphingomyelin from erythrocyte membrane and plasma were increased.