Clinicopathological features of cytokeratin 5-positive pulmonary adenocarcinoma.

Cytokeratin 5 (CK5) is a marker for pulmonary squamous cell carcinoma; however, CK5 is sometimes present in pulmonary adenocarcinoma (ADC), and there is insufficient information regarding the clinicopathological features of CK5-positive ADC. We aimed to explore the clinicopathological characteristics of CK5-positive ADC using immunohistochemistry. We prepared the following two cohorts: a resected cohort containing 220 resected tumours for primarily studying the detailed morphological characteristics, and a tissue microarray (TMA) cohort containing 337 samples for investigating the associations of CK5 expression with other protein expressions, genetic and prognostic findings. CK5-positive ADC was defined to have ≥ 10% tumour cells and presence of CK5-positive tumour cells in the resected and TMA cohorts, respectively. CK5-positive ADCs were identified in 91 (16.3%) patients in the combined cohort. CK5-positive ADCs had male predominance (P = 0.012), smoking history (P = 0.001), higher stage (P < 0.001), histological high-grade components (P < 0.001), vascular invasion (P < 0.001), mucinous differentiation (P < 0.001), spread through airspaces (P < 0.001), EGFR wild-type (P < 0.001), KRAS mutations (P < 0.001), ALK rearrangement (P < 0.001) and ROS1 rearrangement (P = 0.002). In the resected cohort, more than half the CK5-positive ADCs (19 cases, 65.5%) showed mucinous differentiation; the remaining cases harboured high-grade components. In the TMA cohort, CK5-positive ADCs correlated with TTF-1 negativity (P = 0.002) and MUC5B, MUC5AC and HNF4alpha positivity (P < 0.001, 0.048, < 0.001). Further, CK5-positive ADCs had significantly lower disease-free and overall survival rates than CK5-negative ADCs (P < 0.001 for each). Additionally, multivariate analysis revealed that CK5 expression was an independent poor prognostic factor. CK5-positive ADCs showed aggressive clinical behaviour, with high-grade morphology and mucinous differentiation.

Potential egg contamination by Salmonella enterica serovar Typhimurium definitive type 104 following experimental infection of pullets at the onset of lay.

The present study investigated the potential of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) definitive type 104 (DT104) to contaminate eggs in vivo. Of 10 strains of Salmonella Typhimurium DT104, none caused egg contamination when hens were inoculated orally. Three passages of the strains through recovery from the reproductive organs of intravenously infected laying hens resulted in no egg contamination after oral infection of the hens. Feed and water withdrawal for 24 h at 5 and 10 d after oral infection with Salmonella Typhimurium DT104 slightly decreased egg production but did not lead to egg contamination. Finally, oral infection of pullets at the onset of lay (approximately 50% of egg production) resulted in egg contamination (1.7%) in 2 wk. In conclusion, the Salmonella Typhimurium DT104 strains used in the present study have a low possibility of causing egg contamination; however, because infection at the onset of lay can cause egg contamination, the introduction of Salmonella Typhimurium DT104 into the layer houses should be prevented, especially when hens start laying eggs.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test.

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r(2) = 0.95, P < 0.0001) and for diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

[Mediastinal aberrant goiter; report of a case].

A 54-year-old woman was admitted to our hospital because of an abnormal shadow on chest X-ray. Chest computed tomography (CT) scan and magnetic resonance imaging (MRI) demonstrated an anterior mediastinal tumor. The tumor was resected completely through a median sternotomy. The tumor was dissected successfully from the surrounding vessels in spite of the heavy adhesion to them. The blood supply of the tumor was from a branch of the brachiocephalic artery. The tumor was 9 x 8 x 3 cm in size, and was diagnosed as an aberrant mediastinal goiter since it showed no communication to the thyroid gland. An aberrant mediastinal goiter is a quite rare entity of diseases and its removal through the neck would result in uncontrolled blood loss because its blood supply usually derives from intrathoracic vessels.

Imbalanced expression of cellular nuclear oncogenes caused by v-sis/PDGF-2.

Two murine cell lines that overexpress v-sis/PDGF-2 were used to study the mechanism of cell transformation by SSV (simian sarcoma virus). In contrast to the parental cells that are phenotypically normal and serum-dependent for growth, v-sis-overexpressing cells grow in PDGF-free plasma medium, are unable to enter the G0 state and are highly tumorigenic. Analysis of the expression of some growth factor-induced early response genes in v-sis-overexpressing cells revealed: (a) high and constitutive c-myc mRNA levels in SSV-NRK cells; (b) unaltered levels of fra-1, fos B, jun B and krox 20 transcripts; (c) high and constitutive FOS staining due to c-FOS and FOS-related protein(s); (d) constitutive c-JUN and higher JUN D expression. These results are compatible with a model in which endogenous production of v-sis/PDGF-2 leads to deregulated expression of key cellular transregulators that, in turn, alter the cells' transcriptional program leading to the transformed state and malignancy.

fra-2 promoter can respond to serum-stimulation through AP-1 complexes.

fra-2 (fos-related antigen-2) expression is detected at a basal level even in growth-arrested chicken embryo fibroblasts (CEF), but upon serum-stimulation high levels of its transcripts are transiently observed. This induction is delayed and prolonged compared to that of c-fos. Transient expression experiments in CEF using a series of constructs of chicken fra-2 promoter region linked to the CAT reporter gene indicated previously that serum response element (SRE) is not required for full serum inducibility. In this report, we show that constructs in which the CRE-like sequence and both AP-1 binding sites are disrupted lack serum inducibility, suggesting that either of these enhancers is important in serum induction of fra-2. In growth-arrested CEF, small amounts of Fra-2/c-Jun complex bind to the AP-1 consensus sequences in fra-2 promoter, while a significant part of the enhanced AP-1 binding activity after 60-120 min of serum stimulation is attributable to c-Fos/c-Jun heterodimer. At later times Fra-2/c-Jun again becomes the main complex. Transient expression assays in F9 cells indicated that c-Fos/c-Jun heterodimers have strong stimulatory effects on fra-2 promoter activity, while Fra-2/c-Jun complex has lower transcriptional activity than that of c-Jun homodimer. These results suggest that c-Fos (induced at earlier times) and c-Jun proteins are at least partly responsible for serum-induced expression of fra-2.

Cloning and characterisation of the mouse fra-2 gene.

Transcription factor AP-1 is comprised of multiple protein complexes that include members of a family of genes related to the proto-oncogene c-fos. In this report, we have extended the analysis of one member of this family, fos-related antigen-2 (fra-2), by isolating and characterising genomic and cDNA clones encoding the mouse fra-2 homolog. The overall gene structure (number and positions of introns) was similar to that of both the chicken fra-2 gene and other members of the fos family, and the relative positions of putative enhancers in the 5' regulatory region were well conserved between the mouse and chicken fra-2 genes. High levels of fra-2 mRNA were detected in ovary, stomach, small and large intestine, brain, lung and heart. The mouse Fra-2 protein showed 94% and 87.5% conservation with human and chicken Fra-2, respectively, and mouse Fra-2, like the chicken homolog, induced transformation of chicken embryo fibroblasts. The characterisation of the mouse fra-2 gene provides a basis for analysis of Fra-2 function in the whole animal.

Phosphorylation and high level expression of Fra-2 in v-src transformed cells: a pathway of activation of endogenous AP-1.

Chicken embryo fibroblasts (CEF) transformed with v-src were previously reported to revert to normal phenotype after the introduction of dominant-negative mutants of Fos or Jun, indicating that endogenous AP-1 activity is essential for the cellular transformation. The major changes in the expression levels of fos and jun family genes induced by v-src were the elevation of fra-2 and c-jun transcripts. We show here that extensive phosphorylation of the AP-1 component Fra-2 is a major qualitative change in v-src transformed CEF and that several Ser and Thr residues in a C-terminal region of Fra-2 (amino acids 266-323) are phosphorylated specifically. The induced kinase activity was detected at the position of 42 kDa by in gel kinase assay using the Fra-2 C-terminal region as a substrate, and it was identified as chicken ERK2. JNK1 and JNK2, other members of the MAP kinase family, were not significantly activated in v-src transformed CEF and Fra-2 was not a good substrate for JNKs. fra-2 promoter analysis indicated that this promoter activity is elevated in v-src transformed CEF via two AP-1 binding sites and CRE-like sequence. We propose that phosphorylation of Fra-2 by ERK2 converts it from an inefficient transcriptional activator to an active one and further that fra-2 expression is autoregulated in response to the phosphorylation status of its gene product.

Effect of prostaglandin E1 analogue TFC 612 on diabetic neuropathy in streptozocin-induced diabetic rats. Comparison with aldose reductase inhibitor ONO 2235.

The effect of a newly developed oral agent, prostaglandin E1 (PGE1) analogue TFC 612, on diabetic neuropathy was studied by giving it for 6 wk to streptozocin-induced diabetic rats that had been diabetic for 3 mo and was compared with the effects of aldose reductase inhibitor ONO 2235. Although both compounds improved decreased motor nerve conduction velocity, the effect of TFC 612 continued during the 6 wk of treatment, whereas that of ONO 2235 became weaker from wk 4. The abnormality in sciatic nerve sorbitol and myo-inositol levels was reversed with ONO 2235, whereas it was unchanged with TFC 612. With the laser Doppler flowmetry technique, a decrease in the sciatic nerve blood flow in diabetic rats was shown to improve with both compounds, but TFC 612 had a greater effect than ONO 2235, and the increased lactate level of the diabetic nerve was corrected with both compounds, suggesting that both may be associated with the amelioration of ischemia in the diabetic endoneurium. Both TFC 612 and ONO 2235 partially but significantly normalized decreased fiber size in diabetic rats. On the other hand, TFC 612 completely normalized the dilated lumen area in diabetic rats, whereas ONO 2235 did not. These results suggest that the PGE1 analogue TFC 612 has a significant effect on diabetic neuropathy, possibly via vasotropic action, and may be a potent compound for the treatment of diabetic neuropathy.

Amelioration of nerve Na(+)-K(+)-ATPase activity independently of myo-inositol level by PGE1 analogue OP-1206.alpha-CD in streptozocin-induced diabetic rats.

An oral prostaglandin E1 (PGE1) analogue, OP-1206.alpha-CD, was given to rats with streptozocin (STZ)-induced diabetes to examine the therapeutic effects of OP-1206 on short-term and long-term diabetic neuropathy and its action mechanism with special reference to nerve Na(+)-K(+)-ATPase activity. In the short-term experiment, OP-1206 was administered daily to diabetic rats in 3- and 30-mg/kg doses for 4 wk from the day of STZ injection. In the long-term study, 10 micrograms/kg OP-1206 was also given daily for 8 wk from 7 mo after induction of diabetes. The compound improved decreased sciatic motor nerve conduction velocity in both short-term and long-term diabetic rats. The nerve Na(+)-K(+)-ATPase activity of diabetic rats, reduced by 40% compared with controls, was reversed to the level of controls in both experiments, whereas weight loss and hyperglycemia were unchanged, and neither nerve sorbitol accumulation nor myo-inositol depletion was corrected. In a morphometric analysis of myelinated nerve fibers (MNFs) in long-term diabetes, the mean diameter of the largest 10% of MNFs was significantly reduced in untreated diabetic compared with control rats, but OP-1206 completely reversed this reduction. The results suggest that OP-1206 ameliorates a decrease in nerve Na(+)-K(+)-ATPase activity without any effect on nerve myo-inositol level and that the compound may be not only a potent therapeutic agent for the treatment of diabetic neuropathy but also a useful research tool to investigate the mechanism of nerve Na(+)-K(+)-ATPase activity regulation.

Induction of FOS and JUN proteins by adrenocorticotropin and phorbol ester but not by 3',5'-cyclic adenosine monophosphate derivatives.

We report the results of an extensive kinetic analysis of the effects of ACTH, cAMP derivatives (dibutyryl cAMP and 8-bromo-cAMP) and phorbol ester (phorbol-12-myristate-13-acetate) on the expression of fos and jun gene family members at the mRNA (Northern hybridization) and protein levels (immunoprecipitation and indirect immunofluorescence) in the mouse Y-1 adrenocortical cell line. FOS and JUN proteins are induced by ACTH independently of cell cycle stage. c-Fos, fos-B, fra-1, fra-2, c-jun, and jun-B genes are induced by ACTH, the kinetic profiles for mRNAs and respective protein products being similar, except for a 1-h protein delay. Jun-D mRNA is an exception, being constitutively expressed. However, JUN D protein is induced by ACTH. phorbol-12-myristate-13-acetate closely mimics these inductive effects of ACTH. On the other hand, cAMP derivatives are not effective in inducing the fos and jun genes, except for fra-2 mRNA, JUN D protein, and to some extent JUN B protein. Clearly, ACTH is endowed with the versatile capability of modulating fos and jun gene expression, suggesting that AP-1 transcription factors play a role in ACTH mechanisms of action. ACTH receptors are likely to activate signaling routes other than the classical cAMP/protein kinase A in order to induce FOS and JUN proteins.

Biochemical and functional analysis of highly phosphorylated forms of c-Jun protein.

We report here that, upon UV irradiation or growth stimulation, endogenous c-Jun (40 kDa) in chicken embryo fibroblasts (CEF) is converted into several forms with apparently higher molecular weights in SDS-polyacrylamide gel electrophoresis (45, 44, 42 kDa). Two of the bands (44 and 45 kDa) were transient after growth stimulation, but were much more persistent after UV irradiation. In both cases, the drastic mobility shifts were accompanied with the activation of endogenous JNK activity but not of MAPK activity, and the bands were shown to represent different phosphorylation states of c-Jun rather than ubiquitinated c-Jun. Biochemical analysis indicated that phosphorylation at Ser63 and Ser73 was not sufficient to produce these drastic mobility shifts, which additionally required phosphorylation at Thr91 and Thr93. Substitution of both Ser63 and Ser73 with either Ala or Asp had no significant effect on the transforming activity of c-Jun, but the mutants failed to show drastic mobility shifts even after UV irradiation. These results indicate that Ser63 and Ser73 are essential for the drastic mobility shifts and further suggest that the highly phosphorylated forms of c-Jun are not directly involved in cellular transformation.

Asian hereditary neuropathy patients with peripheral myelin protein-22 gene aneuploidy.

Japanese hereditary neuropathy with liability to pressure palsy (HNPP) patients have a deletion of one peripheral myelin protein-22 (PMP22) gene region in distal chromosome band 17p11.2 as do Caucasian patients. Japanese and Asiatic Indian CMT1A patients have a PMP22 gene duplication that results in Charcot-Marie-Tooth disease type IA (CMT1A; HMSNIA) in patients of European and Middle Eastern ancestry. About 70% of Japanese CMT1 patients have a PMP22 duplication as do Caucasians, while Japanese CMT1B, CMT2 and Dejerine-Sottas patients to not have PMP22 gene region aneuploidy. Although HNPP and CMT1A genotypes are generated simultaneously by unequal recombination that results in PMP22 gene aneuploidy in each daughter cell, only 3 Japanese HNPP probands with PMP22 deletion from a large patient population were referred to a single center compared to 18 referred CMT1A probands with PMP22 duplication. This lower HNPP frequency more likely reflects lower HNPP reproductive fitness than patient ascertainment bias because disease severity and variation in severity is about the same in CMT1A and HNPP patients and because all patients of both types were referred regardless of disease severity. These results, along with an apparently high de novo CMT1A mutation rate, suggest that common ancestors of Japanese, Asian Indians, and Caucasians carried PMP22 geneflanking sequences that enhance unequal crossing over.

A case of unilateral diaphragmatic eventration treated by plication with thoracoscopic surgery.

A 56-year-old woman underwent plication with U-stitches by thoracoscopic surgery for left diaphragmatic eventration. Marked improvement in left lung expansion, normalization of the position of the left diaphragm on chest radiograph, and improvement of pulmonary function and dyspnea on exertion have been maintained for 2 years. Plication for diaphragmatic eventration should be performed with minimally invasive surgery.

Synthesis of a molt-inhibiting hormone of the American crayfish, Procambarus clarkii, and determination of the location of its disulfide linkages.

A molt-inhibiting hormone (Prc-MIH) of the American crayfish, Procambarus clarkii, a member of the type II CHH family, was chemically synthesized and the location of its three disulfide linkages was determined. Prc-MIH consists of 75 amino acid residues and was synthesized by a thioester method. Two peptide segments, Boc-[Cys(Acm)(7,24,27), Lys(Boc)(19)]-Prc-MIH(1-39)-SCH(2)CH(2)CO-Nle-NH(2) and H-[Cys(Acm)(40,44,53), Lys(Boc)(42,51,67)]-Prc-MIH(40-75)-NH(2), were prepared using peptides obtained via the Boc solid-phase method. Condensation of the building blocks in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, and N, N-diisopropylethylamine, followed by removal of the protecting groups, gave the reduced form of Prc-MIH(1-75)-NH(2). This product was converted to the native form of Prc-MIH (synthetic Prc-MIH) in a buffer which contained cysteine and cystine. The synthetic Prc-MIH showed the same behavior by RP-HPLC and biological activity assays as the natural Prc-MIH. The disulfide bond between Cys7 and Cys44 was determined by isolation of a fragment from an enzymatic digest of the synthetic Prc-MIH by RP-HPLC, followed by mass analysis. The disulfide bonds between Cys24 and Cys40 and between Cys27 and Cys53 were determined by comparing the elution position of an enzymatic digest of the synthetic Prc-MIH with authentic chemically synthesized samples, which contained three types of possible disulfide linkages.

A combination of the aldose reductase inhibitor, statil, and the prostaglandin E1 analogue, OP1206.alpha CD, completely improves sciatic motor nerve conduction velocity in streptozocin-induced chronically diabetic rats.

In view of the possible implication of multifactorial mechanisms in the pathogenesis of diabetic neuropathy, the aldose reductase inhibitor (ARI), Statil, which ameliorates abnormal sorbitol or myo-inositol metabolism in diabetic nerves, and the prostaglandin E1 (PGE1) analogue, OP1206.alpha CD (OP), which improves diabetic vascular derangements, were administered simultaneously for 2 months to streptozocin (STZ)-induced diabetic rats with 5 months' duration of diabetes, and the effects on sciatic motor nerve conduction velocity (MNCV), Na(+)-K(+)-adenosine triphosphatase (ATPase) activity, and morphology of myelinated nerve fibers (MNF) were compared with the effects of a monotherapy with OP. The combination regimen ameliorated abnormal nerve sorbitol and myo-inositol levels and normalized decreased MNCV and enzyme activity. In contrast, neither sorbitol nor myo-inositol metabolism was ameliorated, and only insufficient improvement of MNCV and morphology of MNF was obtained with a monotherapy with OP. In addition, the combination therapy reversed both a decrease in the percent of large MNF and an increase in the percent of small MNF in diabetic rats, whereas a monotherapy with OP reversed only a decrease in the percent of large MNF. The results might suggest that a multiple-drug therapy with different mechanisms of action has greater effects on diabetic neuropathy than a single-drug therapy and is worthy of clinical consideration.

Phosphotyrosine of macrophage by low-density lipoproteins from hemodialysis patients.

BACKGROUND: Because of the possible importance of tyrosine phosphorylation in the signal transduction process, we investigated whether an interaction of low-density lipoprotein (LDL) from hemodialysis patients (HD-LDL) and human macrophages induces tyrosine-phosphorylated proteins in the macrophages. METHODS: Human monocyte-derived macrophages were incubated with HD-LDL (100 micrograms/ml) or native LDL (100 micrograms/ml) for 15 minutes at 37 degrees C. Whole cells were lyzed with Tris-HCl buffer containing vanadate and Triton X-100. After centrifugation, lyzed proteins were divided into Triton-soluble and -insoluble fractions. Both fractions (soluble and insoluble) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Immunoblotting was performed using an antibody against phosphotyrosine or c-Src. RESULTS: Several proteins in the range 40 to 100 kDa were found to be phosphorylated constitutively in the macrophages and not affected by the addition of HD-LDL. HD-LDL did not induce any tyrosine-phosphorylated proteins either in the soluble or insoluble fractions. Macrophages pretreated with tyrosine kinase inhibitor genestein drastically inhibited tyrosine phosphorylation of these proteins. The nonreceptor tyrosine kinase, c-Src p60, was also strongly tyrosine phosphorylated in the macrophages, and this was not enhanced by the stimulation of HD-LDL. CONCLUSION: These data suggest that tyrosine autophosphorylated proteins may play a role in the early step of signal transduction in the macrophages.

Continuous recording of cerebral blood flow by means of a thermal diffusion method using a Peltier stack.

The thermal diffusion method is known to be effective for making quantitative measurements of blood flow, but cannot be easily applied to problems concerning quantitative changes in blood flow. Carter et al. found that the thermal diffusion technique using a Peltier stack as the probe produced extremely stable recordings and was suitable for quantitative work. We made a more stable probe containing an air space and having a stainless cap that added weight. A stable recording of blood flow was then possible. For calibration of the probe, we used blood flow values measured by means of an electrolytic technique and the equation proposed by Carter et al. In the present study, we have shown that it is theoretically possible to perform the calibration even without obtaining data on blood flow after cardiac arrest. Experimentally, the validity of such calibration was confirmed. This technique for measuring blood flow should be applicable in various fields and its use in a clinical setting, particularly in the monitoring of blood flow during neurosurgical operations, can be expected.

Morphological characteristics of dermal diabetic microangiopathy.

In an attempt to characterize dermal diabetic microangiopathy, dermal microvessels were qualitatively and quantitatively assessed in 26 healthy control subjects and 47 non-insulin-dependent diabetic patients. Basement membrane thickness (BMT) was significantly greater and the homogeneous BM more frequent in diabetic than in control microvessels. BMT did not change with duration of diabetes but significantly decreased with age and onset age of diabetes, suggesting either a different process of BM thickening with respect to age in diabetic dermal microvessels or the implication of genetic influence on the pathogenesis of dermal diabetic microangiopathy. Endothelial cell hyperplasia was not noted in dermal diabetic microvessels. In view of the fact that the hyperplasia has been reported in other tissues and is closely associated with diabetic microangiopathy, the nature of dermal microvessels or endothelial cells or the milieu around dermal microvessels might be different from other organs.

Multiple intracranial aneurysms associated with moyamoya disease. Case report.

A case of multiple intracranial aneurysms associated with unilateral moyamoya vessels is reported. The authors have reviewed the age, sex, initial symptoms, site of aneurysm, and operative indication in similar cases reported in the literature. It was found that aneurysms associated with moyamoya disease were frequently located in the vertebrobasilar system.