Use of immunoblotting to detect Aspergillus fumigatus antigen in sera and urines of rats with experimental invasive aspergillosis.

Immunoblotting was used to detect Aspergillus fumigatus antigen in sera and urines of immunosuppressed rats experimentally infected with A. fumigatus. Organisms were administered by both intravenous and intratracheal injections. Intravenously infected rats developed disseminated aspergillosis, but intratracheally infected rats developed pulmonary disease only. Fungal cultures of blood and urine samples from infected rats were negative. In the urines of intravenously infected rats, antigen was detected 24 to 48 h after infection; in the urines of intratracheally infected animals, antigen was detected on days 4 to 5 after infection. Antigen in serum was detected later than antigen in urine was. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of serum and urine samples, the most strongly reacting antigenic materials were found in the 88-, 40-, 27-, and 20-kilodalton regions. These dominant antigens appeared to be the same as those of control antigens prepared from A. fumigatus grown in vitro. Rabbit antiserum to Aspergillus filtrate antigen was found to be more immunoreactive than antiserum to mycelial or conidial antigen. No mycelium-specific antigens were detected.     

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.     

Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas

Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. Cox-2 is an induced isoform of cyclooxygenases that plays an important role in carcinogenesis, suppression of apoptosis, angiogenesis, and metastasis of colon cancer. We report frequent co-expression of cox-2 and laminin-5 at the invasive front of early-stage lung adenocarcinomas. We investigated the expression of cox-2 and laminin-5 immunohistochemically in 102 cases of small-sized lung adenocarcinoma (maximum dimension, 2 cm or less). Cox-2 and laminin-5 were expressed in 97 (95.1%) and 82 (80.4%) cases, respectively. Both were preferentially localized in cancer cells at the cancer-stroma interface, although cox-2 tended to show a diffuse staining pattern in some cases. A comparison of their staining patterns revealed a striking similarity in their distribution in 24 cases, and a partial overlap between their localization in another 20 cases. Moreover, an overall correlation was found between the expression levels of cox-2 and laminin-5 (P = 0.018). To gain insight into the mechanisms that regulate the expression of these proteins, we additionally studied their expression in 58 cases of stage I lung adenocarcinoma, in which p53 status was determined by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism analysis, and direct sequencing. The results showed that tumors with mutant p53 tended to express more cox-2 than those with wild-type p53 (P = 0.080). Also, tumors that overexpressed p53 had higher levels of cox-2 and laminin-5 than those without p53 overexpression (P = 0.032 and 0.047, respectively). Further immunohistochemical analysis showed that tumors that overexpressed both epidermal growth factor receptor (EGFR) and erbB-2 had higher levels of cox-2 and laminin-5 than those without concomitant overexpression of these proteins (P = 0.014 and P = 0.018, respectively). To see whether EGFR signaling is involved in cox-2 and laminin-5 expression, we further conducted in vitro analyses using six lung adenocarcinoma cell lines (A549, HLC-1, ABC-1, LC-2/ad, VMRC-LCD, and L27). Western blot analyses showed that cox-2 mRNA levels, and to a lesser extent laminin-5 gamma2 mRNA levels, correlated with the expression levels of erbB-2 and the phosphorylated form of MAPK/ERK-1/2 protein. The addition of transforming growth factor-alpha increased both cox-2 and laminin-5 gamma2 mRNA levels in A549, ABC-1, and L27 with different kinetics; the induction of cox-2 occurred earlier than that of laminin-5 gamma2. Finally, the migration of ABC-1 cells was inhibited by MAP kinase kinase inhibitor PD98059 and a selective cox-2 inhibitor NS-398. In contrast, the migration of A549 cells was inhibited by PD98059, but much less effectively by NS-398. These results suggest that co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5 at the invasive front of lung adenocarcinomas and that EGFR signaling could be one of the mechanisms. Further investigations are warranted concerning the role of cox-2 and laminin-5 in cancer cell invasion and the significance of p53 and EGFR signaling in the regulation of cox-2 and laminin-5 expression.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Ontogenetic transition in fetal wound transforming growth factor-beta regulation correlates with collagen organization

Fetal rat skin transitions from scarless fetal-type repair to adult-type repair with scar between day 16 (E16) and day 18 (E18) of gestation (term = 21.5 days). Deficient transforming growth factor (TGF)-beta 1 and -beta 2 injury response has been proposed as a mechanism for scarless fetal-type repair. However, previous fetal studies have inconsistently reported the degree of TGF-beta induction after injury. To minimize developmental variables in fetal versus adult TGF-beta regulation, we narrowed our study to wounded fetal animals. We hypothesize that TGF-beta ligand and receptor expression will be differentially regulated during the transition from early gestation (E16) wounds manifesting scarless fetal-type repair to late gestation (E19) wounds manifesting adult-type repair with scar. In this study, decreased and rapidly cleared TGF-beta 1 and -beta 2 expression accompanied by increased and prolonged TGF-beta 3 levels in wounded E16 animals correlated with organized collagen deposition. In contrast, increased and prolonged TGF-beta 1 and -beta 2 expression accompanied by decreased and delayed TGF-beta 3 expression in wounded E19 animals correlated with disorganized collagen architecture. Similarly, expression of TGF-beta receptors type I and II were also increased or prolonged in E19 animals. Our results implicate increased TGF-beta 1, -beta 2, and decreased TGF-beta 3 expression, as well as increased type I and II receptor expression in late gestation fetal scar formation.     

Expression of vascular endothelial growth factor receptor 3 in blood and lymphatic vessels of lung adenocarcinoma

Vascular endothelial growth factor receptor 3 (VEGFR-3) has been proposed as a marker for lymphatic endothelial cells. This study investigated the expression of VEGFR-3 in the tumour vessels of lung adenocarcinoma and evaluated whether VEGFR-3 staining was useful for identifying lymphatic vessels within the tumour stroma. It also explored whether active growth of lymphatic vessels occurred in lung adenocarcinoma. Formalin-fixed, paraffin-embedded specimens obtained from 60 cases of lung adenocarcinoma, including five cases of pure bronchiolo-alveolar carcinoma (BAC) without stromal, vascular, and pleural invasion, were examined. No VEGFR-3-positive vessels were observed in pure BAC, but varying numbers of VEGFR-3-positive vessels were found in 39 of 55 (70.9%) invasive adenocarcinomas. A comparison of serial sections stained for VEGFR-3, CD31, and laminin-1 showed that most of the VEGFR-3-positive vessels appeared to be blood vessels (CD31-positive, laminin-1-positive), but some had the characteristics of lymphatic vessels (variable staining for CD31, little or no staining for laminin-1). VEGFR-3 staining highlighted lymphatic invasion by cancer cells; this invasion could not be detected by CD31 or haematoxylin and eosin (H&E) staining. Active growth of lymphatic vessels (as indicated by nuclear Ki-67 labelling of the endothelium) was observed in five tumours, four of which showed a high level of lymphatic invasion by cancer cells. It was concluded that VEGFR-3 immunostaining did not discriminate clearly between vascular and lymphatic endothelial cells, since expression of VEGFR-3 can be up-regulated in tumour blood vessels. However, VEGFR-3 staining combined with laminin-1 and CD31 staining would be useful for identifying lymphatic vessels and their invasion by tumour cells in a more objective way. Finally, proliferation of lymphatic endothelial cells may occur in association with lymphatic invasion by cancer cells.     

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.     

Presumed inhibitory neurons in the macaque inferior temporal cortex: visual response properties and functional interactions with adjacent neurons

Neurons in area TE of the monkey inferior temporal cortex respond selectively to images of particular objects or their characteristic visual features. The mechanism of generation of the stimulus selectivity, however, is largely unknown. This study addresses the role of inhibitory TE neurons in this process by examining their visual response properties and interactions with adjacent target neurons. We applied cross-correlation analysis to spike trains simultaneously recorded from pairs of adjacent neurons in anesthetized macaques. Neurons whose activity preceded a decrease in activity from their partner were presumed to be inhibitory neurons. Excitatory neurons were also identified as the source neuron of excitatory linkage as evidenced by a sharp peak displaced from the 0-ms bin in cross-correlograms. Most inhibitory neurons responded to a variety of visual stimuli in our stimulus set, which consisted of several dozen geometrical figures and photographs of objects, with a clear stimulus preference. On average, 10% of the stimuli increased firing rates of the inhibitory neurons. Both excitatory and inhibitory neurons exhibited a similar degree of stimulus selectivity. Although inhibitory neurons occasionally shared the most preferred stimuli with their target neurons, overall stimulus preferences were less similar between adjacent neurons with inhibitory linkages than adjacent neurons with common inputs and/or excitatory linkages. These results suggest that inhibitory neurons in area TE are activated selectively and exert stimulus-specific inhibition on adjacent neurons, contributing to shaping of stimulus selectivity of TE neurons.