Method for In Situ Evaluation of Superoxide Production by Pulmonary Macrophages in the Rat

In order to investigate superoxide production by pulmonary macrophages in the rat, a route was created by ligating both the inferior and superior venae cavae and resecting the aorta after cannulation through the inferior vena cava into the right atrium of the heart. Lung perfusion was performed via this route with nitro blue tetrazolium. Although there was no formazan deposition throughout the lung, it became detectable in both alveolar and interstitial macrophages when phorbol myristate acetate was added to the perfusate. This deposition was markedly enhanced by previous injection of Corynebacterium parvum. The deposition disappeared after further addition of Cu(Lys)₂, a scavenger of superoxide anions. This procedure may be useful for estimating in situ the ability of pulmonary macrophages to produce superoxide in the rat.     

Phosphorylation of Ser-3 of cofilin regulates its essential function on actin

Background: Cofilin is a low-molecular weight actin-modulating protein, and is structurally and functionally conserved in eucaryotes from yeast to mammals. The functions of cofilin appear to be regulated by phosphorylation and dephosphorylation. Results: A proteolytic study of phosphorylated porcine cofilin and expression of a mutated cofilin in cultured cells revealed that Ser-3 is the unique phosphorylation site. Phosphorylated cofilin was found not to bind to either F-or G-actin while unphosphorylated cofilin binds to both. S3D-cofilin, in which Ser-3 was replaced with Asp, did not bind in vitro to actin while S3A-cofilin did. The transient overexpression of wild-type or S3A-cofilin in cultured cells caused disruption of pre-existing actin structures and induced cytoplasmic actin bundles. Heat shock-induced nuclear or NaCl buffer-induced cytoplasmic actin/cofilin rods contained the expressed cofilin. In contrast, the overexpression of S3D-cofilin did not alter the actin structures. Induced actin rods did not contain S3D-cofilin. S3D-porcine cofilin did not complement the lethality associated with Δcof1 mutations in Saccharomyces cerevisiae while wild-type and S3A-cofilin did. Furthermore, we found that S2A/S4D- and S2D/S4D-yeast cofilin mutants were not viable. Conclusion: We conclude that the function of cofilin is negatively regulated in vivo by phosphorylation of Ser-3 and that cells require the functions of unphosphorylated cofilin for viability.     

Evidence for effector independent and dependent representations and their differential time course of acquisition during motor sequence learning

To investigate the representation of motor sequence, we tested transfer effects in a motor sequence learning paradigm. We hypothesize that there are two sequence representations, effector independent and dependent. Further, we postulate that the effector independent representation is in visual/spatial coordinates, that the effector dependent representation is in motor coordinates, and that their time courses of acquisition during learning are different. Twelve subjects were tested in a modified 2x10 task. Subjects learned to press two keys (called a set) successively on a keypad in response to two lighted squares on a 3x3 display. The complete sequence to be learned was composed of ten such sets, called a hyperset. Training was given in the normal condition and sequence recall was assessed in the early, intermediate, and late stages in three conditions, normal, visual, and motor. In the visual condition, finger-keypad mapping was rotated 90 degrees while the keypad-display mapping was kept identical to normal. In the motor condition, the keypad-display mapping was also rotated 90 degrees, resulting in an identical finger-display mapping as in normal. Subjects formed two groups with each group using a different normal condition. One group learned the sequence in a standard keypad-hand setting and subsequently recalled the sequence using a rotated keypad-hand setting in the test conditions. The second group learned the sequence with a rotated keypad-hand setting and subsequently recalled the sequence with a standard keypad-hand setting in the test conditions. Response time (RT) and sequencing errors during recall were recorded. Although subjects committed more sequencing errors in both testing conditions, visual and motor, as compared to the normal condition, the errors were below chance level. Sequencing errors did not differ significantly between visual and motor conditions. Further, the sequence recall accuracy was over 70% even by the early stage when the subjects performed the sequence for the first time with the altered conditions, visual and motor. There were parallel improvements thereafter in all the conditions. These results of positive transfer of sequence knowledge across conditions that use dissimilar finger movements point to an effector independent sequence representation, possibly in visual/spatial coordinates. Initially the RTs were similar in the visual and the motor conditions, but with training RTs in the motor condition became significantly shorter than in the visual condition, as revealed by significant interaction for the testing stage and condition term in the repeated measures ANOVA. Moreover, using RTs for single key pressing in the three conditions as baseline indices, it was again observed that RTs in the visual and motor conditions were not significantly different in the early stage, but motor RTs became significantly shorter by the late testing stage. These results support the hypothesis that the motor condition benefits more than the visual because it uses identical effector movements to the normal condition. Further, these results argue for the existence of effector dependent sequence representation, in motor coordinates, which is acquired relatively slowly. The difference in the time course of learning of these two representations may account for the differential involvement of brain areas in early and late learning phases found in lesion and imaging studies.     

Sequence analysis of two serological variants, HLA-A2K and HLA-A9HH, detected in Japanese

Two rare variants of HLA-A locus antigens, tentatively called HLA-A2K and HLA-A9HH, were serologically identified in the Japanese population. A2K and A9HH showed short reaction patterns of a series of anti-A2 and anti-A9 sera, respectively. The latter variant also reacted with some anti-A2 sera. Nucleotide sequences of full-length cDNAs for A2K and A9HH were determined. The results revealed that both antigens are encoded by previously undescribed alleles. The nucleotide sequence of the allele for A2K was identical to that of A^*0207 except for a single nucleotide difference in exon 3. The nucleotide sequence of the allele for A9HH was identical to that of A^*2402 except for two nucleotides in exon 2. These two nucleotides are shared by all the reported A2 alleles. These sequencing results the allele for A9HH were consistent with the serological cross-reactivity of A9HH with some anti-A2 sera.     

Biological properties and gene expression associated with metastatic potential of human osteosarcoma

Lung metastasis has a great influence on the prognosis of patients with osteosarcoma. We previously established two high-metastatic sublines, M112 and M132, from the HuO9 human osteosarcoma cell line by in vivo selection. In this study, we newly isolated a high-metastatic subline, H3, and three low-metastatic sublines, L6, L12 and L13, from HuO9 by the dilution plating method. Three high-metastatic sublines produced more than 200 metastatic nodules in the lung, while three low-metastatic sublines produced no or few nodules after injection of 2 × 10⁶ cells into the tail vein of nude mice. There were significant differences in the motility and invasiveness between high- and low-metastatic sublines, whereas the growth rates in vitro and the tumorigenicity in vivo showed no correlation with their metastatic abilities. Early adherence to culture plates was significantly lower in two of three low-metastatic sublines, which occupied smaller surface areas on the culture plates than other sublines did. Comparison of the expression of 637 cancer-related genes by cDNA microarray revealed that seven genes were differentially expressed between high- and low-metastatic sublines. Among them, five genes (AXL, TGFA, COLL7A1, WNT5A, and MKK6) were associated with adherence, motility, and/or invasiveness. These results suggest that the differences in motility/invasiveness and adhesive abilities are key determinants of lung metastasis in osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a rapid PCR method using the insertion sequence IS1203 for genotyping Shiga toxin-producing Escherichia coli O157

We developed a rapid PCR method utilizing the diversity of the insertion site IS1203 for genotyping Shiga toxin-producing Escherichia coli (STEC) O157 (IS1203 PCR typing). DNA fragments digested by PvuII, which cut IS1203 at one site, were ligated with themselves and detected by PCR with outward-facing primer pairs for IS1203. To minimize nonspecific bands, nested PCR was also performed. Two fingerprinting patterns produced from the upstream or downstream regions of IS1203 were obtained within 1 or 2 days. By combining the two patterns, 79 STEC O157 isolates were classified into 39 types, which were then classified into 36 subtypes by pulsed-field gel electrophoresis (PFGE). The discriminatory power of IS1203 PCR typing (D = 0.974) is similar to that of PFGE (D = 0.981). This method can be used for rapid and simplified genotyping.     

Percutaneous transluminal angioplasty against arteriosclerosis obliterans in dialysis patients

The incidence of peripheral arteriosclerosis is on the increase in chronic hemodialysis patients. Recently, the intervention (IV) treatment is conducted to deal with this problem. IV was performed in 4 dialysis patients against the complication of arteriosclerosis obliterans (ASO) but the result was unsuccessful in 3 of them. These 3 failure cases were investigated to find the problems associated with percutaneous transluminal angioplasty (PTA). Cases 1, 2 and 3 had intermittent claudication while case 4 had gangrenous toes as the major complaint. The symptoms in these cases were attributable to 90-100% stenosis and occlusion of superficial femoral artery, bilateral iliac arteries, bilateral superficial femoral-popliteal artery, branch of right iliac artery and left iliac artery region, respectively. IV was successful in case 1 but failed in cases 2 and 4 because the catheter itself did not go through due to the severe stenosis of vessel or the procedure of forcefully dilating the vessel caused dispersion of minute thrombi. In case 3, acute myocardial infarction occurred at 10 h after successful IV, resulting in sudden death. In view of the extent of invasion, IV is a treatment method selected against ASO in dialysis patients. However, the method has a high risk of causing thrombus formation, vessel rupture and organ failure. In this regard, it is advisable to evaluate the systemic condition and conduct IV if the extent of stenosis is mild.     

Differential expression of binding sites for chemokine RANTES on human T lymphocytes

The C-C chemokine RANTES, a T lymphocyte chemoattractant, is considered an important mediator of inflammation, allergy, and host defense against HIV-1 infection. In this study, we investigated the modulation of binding of RANTES to T lymphocytes. Human peripheral blood CD3+ T cells, when freshly isolated from buffy-coat blood, expressed a considerable number of high-affinity binding sites for RANTES. These cells also showed significant chemotactic migration in response to RANTES in vitro. After 6–15 h incubation at 37°C, the binding of RANTES, but not of macrophage inflammatory protein-1α (MIP-1α) or of monocyte chemotactic protein-3 (MCP-3), consistently increased. Scatchard analyses indicated that the number of binding sites for RANTES increased about threefold by 15 h without any change in the affinity. The increase in RANTES binding was no longer detected by 24 h. This increase in the specific binding was mainly attributable to CD4⁺ T cells and was not associated with increased chemotactic activity of these cells in response to RANTES. Incubation with anti-CD3 antibody for 15 h markedly reduced the binding capability of T cells for RANTES and was associated with decreased chemotactic activity. On the other hand, when T cells were incubated with interleukin-2 (IL-2) for 1 week, the specific binding for all three C-C chemokines, RANTES, MIP-1α, and MCP-3 was markedly increased in comparison to cells cultured in the absence of IL-2. These results suggest that the expression of binding sites on T cells for RANTES is differentially modulated, indicating the existence of novel receptors for RANTES that do not bind MIP-1α.     

A girl with 1p36 deletion syndrome and congenital fiber type disproportion myopathy

Chromosome 1p36 deletion syndrome is characterized by hypotonia, moderate to severe developmental and growth retardation, and characteristic craniofacial dysmorphism. Muscle hypotonia and delayed motor development are almost constant features of the syndrome. We report a 4-year-old Japanese girl with 1p36 deletion syndrome whose muscle pathology showed congenital fiber type disproportion (CFTD) myopathy. This is the first case report of 1p36 deletion associated with CFTD. This association may indicate that one of the CFTD loci is located at 1p36. Ski proto-oncogene −/− mice have phenotypes that resemble some of the features observed in patients with 1p36 deletion syndrome. Because fluorescent in situ hybridization analysis revealed that the human SKI gene is deleted in our patient, some genes in 1p36, including SKI proto-oncogene, may be involved in muscle hypotonia and delayed motor development in this syndrome. Received: March 4, 2002 / Accepted: July 7, 2002     

Lung adenocarcinoma with bronchioloalveolar carcinoma component is frequently associated with foci of high-grade atypical adenomatous hyperplasia

We assessed the occurrence of atypical adenomatous hyperplasia (AAH) in whole lung lobes with primary cancer lesions. Following surgical resection, tissue specimens were sliced to a thickness of 4 mm (3,641 specimens from 61 cases; mean = 59.7 specimens per case). A total of 119 AAH foci were found and an association was evident in 25 (57%) of 44 adenocarcinomas, 3 (30%) of 10 squamous cell carcinomas, and 2 (29%) of 7 other lung cancers. Histologic evaluation showed that 108 AAH foci were categorized as low-grade and the other 11 as high-grade AAH. These 11 foci of high-grade AAH were present in 7 patients with adenocarcinoma, and in 1 patient there was a synchronous double primary lung adenocarcinoma. High-grade AAH was closely associated with bronchioloalveolar carcinoma (BAC) type adenocarcinoma, and low-grade AAH with non-BAC adenocarcinoma. The mean +/- SD Ki-67 labeling index in high-grade AAH (3.5%+/-2.9%) was significantly higher than for the low-grade index (1.4%+/-1.6%). We propose that foci of high- but not low-grade AAH may be potential precursor lesions of lung adenocarcinoma, especially with the BAC component.