IL-6-Producing Pheochromocytoma Associated With Von Hippel Lindau Disease: A Case Report With Literature Review.

We present a first case report of an IL-6-producing pheochromocytoma associated with von Hippel Lindau (vHL) disease. Pheochromocytomas are rare tumors that produce catecholamines, leading to various symptoms. In this case, a 28-year-old woman with a family history of vHL disease presented with a prolonged fever. Laboratory examinations revealed elevated C-reactive protein levels, and notably, a significantly increased serum IL-6 level. Imaging studies confirmed bilateral adrenal tumors with increased uptake on fluorodeoxyglucose-positron emission tomography and 123I-metaiodobenzylguanidine scintigraphy in the right adrenal gland. Despite partial relief with nonsteroidal anti-inflammatory drugs and alpha-blockers, her fever persisted until prednisolone administration, which promoted a complete resolution. A histopathological analysis following a right laparoscopic adrenalectomy revealed a typical pheochromocytoma. We conducted further analyses, including an enzyme-linked immunosorbent assay (ELISA), a quantitative real-time polymerase chain reaction (PCR) test, and immunoblot assays from the resected tumor tissues. We compared the current case with other cases of pheochromocytoma that presented neither elevated serum IL-6 nor high fever. Using ELISA, we found that this patient exhibited more IL-6 secretion than that seen in other cases. Additionally, quantitative real-time PCR and immunoblot found that both the phosphorylated signal transducer and activator of transcription 3 (STAT3) messenger RNA (mRNA) and protein expression levels exceeded those of the other cases. Thus, we surmised that IL-6 was produced directly from the tumor tissue and IL-6 expression was potentiated through the IL-6/STAT3 signaling pathway. Our findings contribute to the understanding of IL-6-producing pheochromocytomas and their distinct clinical characteristics.

Contrast-enhanced intraoperative ultrasonography using perfluorobutane microbubbles for the enumeration of colorectal liver metastases.

BACKGROUND: Intraoperative ultrasonography (IOUS) is considered the standard for the identification of liver metastases. Use of lipid-stabilized perfluorobutane microbubbles as an ultrasound contrast agent may improve this. The value of contrast-enhanced IOUS (CE-IOUS) in enumerating colorectal liver metastases was studied here. METHODS: CE-IOUS was performed in consecutive resections for colorectal liver metastases in 2007-2010. All patients underwent preoperative computed tomography. Magnetic resonance imaging was not carried out routinely. Conventional intraoperative examination including IOUS, and CE-IOUS with peripherally injected contrast were performed. The histopathological findings and 6-month follow-up images were used as the reference standard. RESULTS: The study group of 102 patients had a total of 315 lesions identified on preoperative imaging (2·4 lesions per operation; 129 operations). Conventional intraoperative examination including IOUS identified 350 lesions (2·7 per operation). CE-IOUS identified 370 lesions (2·9 per operation). The sensitivity, specificity and accuracy of CE-IOUS were 97·1, 59·1 and 93·2 per cent respectively. The CE-IOUS findings altered the surgical plan in 19 operations (14·7 per cent). CONCLUSION: CE-IOUS provided additional information to that obtained using contemporary preoperative imaging and conventional intraoperative examinations.

Volume imaging by tracking sparse topological features in electron micrograph tilt series.

The sensitive coherent interference of electron waves arising from a specimen is useful for revealing subtle structural information in electron micrographs, which can be important for minimising dose and for rapid imaging. In general, dynamical diffraction is expected due to the useful strong interactions of electrons with matter, which can create phase contrast that violates the requisite Radon projection assumption for tomography. It is for these reasons that incoherent imaging modalities such as high angle annular dark field have been favoured to date in electron tomography of crystalline specimens, to access a monotonic relationship between specimen thickness and micrograph intensity. Here we use a geometric approach to track topological features that are robust to perturbation of the imaging conditions, to enable 3D reconstructions from electron microscope tilt series under imaging conditions that violate the Radon projection assumption, with an emphasis on phase contrast. Invoking a sparsity assumption, we demonstrate that topological features can be reliably tracked in 3D using a differential geometric form of stereoscopy, to circumvent departures from the projection approximation and reduce noise by effecting segmentation of interest points from the outset. We demonstrate this approach on a variety of different specimen and data types, from polyhedral nanoparticles, to steel dislocation networks, cryo-EM cellular structures and 3D diffuse diffraction of a relaxor ferroelectric.

Suppression of developmental anomalies by maternal macrophages in mice.

We tested whether nonspecific tumoricidal immune cells can suppress congenital malformations by killing precursor cells destined to cause such defects. Pretreatment of pregnant ICR mice with synthetic (Pyran copolymer) and biological (Bacillus Calmette-Guérin) agents significantly suppressed radiation- and chemical-induced congenital malformations (cleft palate, digit anomalies, tail anomalies, etc.). Such suppressive effects were associated with the activation of maternal macrophages by these agents, but were lost either after the disruption of activated macrophages by supersonic waves or by inhibition of their lysosomal enzyme activity with trypan blue. These results indicate that a live activated macrophage with active lysosomal enzymes can be an effector cell to suppress maldevelopment. A similar reduction by activated macrophages was observed in strain CL/Fr, which has a high spontaneous frequency of cleft lips and palates. Furthermore, Pyran-activated maternal macrophages could pass through the placenta, and enhanced urethane-induced cell killing (but not somatic mutation) in the embryo. It is likely that a maternal immunosurveillance system eliminating preteratogenic cells allows for the replacement with normal totipotent blast cells during the pregnancy to protect abnormal development.

Diversity and organization of human T cell receptor delta variable gene segments.

Previous studies of the human TCR-delta gene identified a single commonly used V delta segment, denoted V delta 1. To better understand the extent of the human TCR-delta V gene repertoire, TCR-delta transcripts and gene rearrangements were examined in a new panel of cloned human TCR-gamma/delta lymphocytes. Through this analysis we identified and determined the structures of two new V delta segments, denoted V delta 2 and V delta 3. These V delta segments are different from previously characterized V alpha segments, supporting the notion that the human V delta and V alpha repertoires are distinct. Examination of V gamma gene segment usage in these cells reveals that the V delta 2 gene segment is used in conjunction with the V gamma 2 gene segment. Blot hybridization indicates that the V delta 2 gene segment lies between V delta 1 and D delta-J delta-C delta, and within 100 kb of the latter. Analysis of genomic clones indicates that the V delta 3 gene segment lies in an inverted orientation, approximately 2 kb 3' of C delta. This implies that rearrangement of V delta 3 to D delta-J delta-C delta occurs by inversion. Together with previous mapping studies, these results indicate that human V delta segments are dispersed, rather than clustered, within the TCR-alpha/delta locus. The analysis of rearrangements in polyclonal thymocyte DNA suggests that there may be a limited number of additional V delta gene segments yet to be characterized.

Significance of collagenous and mucinous spherulosis in breast cytology specimens.

OBJECTIVE: Spherulosis of the breast is a rare but distinct benign morphological entity. As there are few cytological reports of breast spherulosis, the significance of spherulosis among cytological specimens is unclear. The objective was to document cytological aspects of spherulosis. METHODS: A total of 3491 consecutive breast fine needle aspiration cytology (FNAC) samples and 69 nipple discharge cytology samples were reviewed. Papanicolaou-stained slides with or without Romanowsky staining were analysed. The corresponding 1926 histological specimens were also reviewed. RESULTS: We detected 17 cases of collagenous spherulosis (CS) and/or mucinous spherulosis (MS) among 3560 breast cytology specimens (0.48%). All samples were from women, who varied in age from 22 to 69 years. CS and/or MS were present in 15 of 3491 FNAC specimens (0.43%) and in two of 69 nipple discharge cytology specimens (2.9%). Corresponding histological specimens were available for 14 of the 17 specimens. Of the 14 specimens, 12 consisted of intraductal papilloma, one of fibroadenoma, and one of fibrocystic change. There was no spherulosis among the 1251 cytological specimens of malignant diseases. CONCLUSIONS: Cytological evidence of spherulosis is a good indicator of intraductal papilloma.

Identification of putative human T cell receptor delta complementary DNA clones.

A novel T cell receptor (TCR) subunit termed TCR delta, associated with TCR gamma and CD3 polypeptides, was recently found on a subpopulation of human T lymphocytes. T cell-specific complementary DNA clones present in a human TCR gamma delta T cell complementary DNA library were obtained and characterized in order to identify candidate clones encoding TCR delta. One cross-hybridizing group of clones detected transcripts that are expressed in lymphocytes bearing TCR gamma delta but not in other T lymphocytes and are encoded by genes that are rearranged in TCR gamma delta lymphocytes but deleted in other T lymphocytes. Their sequences indicate homology to the variable, joining, and constant elements of other TCR and immunoglobulin genes. These characteristics, as well as the immunochemical data presented in a companion paper, are strong evidence that the complementary DNA clones encode TCR delta.

A T cell-specific transcriptional enhancer within the human T cell receptor delta locus.

The T cell antigen receptor (TCR) delta gene is located within the TCR alpha locus. A T cell-specific transcriptional enhancer, distinct from the TCR alpha enhancer, has been identified within the J delta 3-C delta intron of the human T cell receptor delta gene. This enhancer activates transcription from the V delta 1 and V delta 3 promoters as well as from heterologous promoters. Enhancer activity has been localized to a 250-bp region that contains multiple binding sites for nuclear proteins. Thus, transcriptional control of the TCR delta and TCR alpha genes is mediated by distinct regulatory elements.

Immunochemical proof that a novel rearranging gene encodes the T cell receptor delta subunit.

The T cell receptor (TCR) delta protein is expressed as part of a heterodimer with TCR gamma, in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR delta was produced that binds specifically to the surface of several TCR gamma delta cell lines and immunoprecipitates the TCR gamma delta as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR delta subunit alone after chain separation. A candidate human TCR delta complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR gamma delta cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR delta. This complementary DNA clone thus corresponds to the gene that encodes the TCR delta subunit.

Structurally divergent human T cell receptor gamma proteins encoded by distinct C gamma genes.

The human T cell receptor (TCR) gamma polypeptide occurs in structurally distinct forms on certain peripheral blood T lymphocytes. Complementary DNA clones representing the transcripts of functionally rearranged TCR gamma genes in these cells have been analyzed. The expression of a disulfide-linked and a nondisulfide-linked form of TCR gamma correlates with the use of the C gamma 1 and C gamma 2 constant-region gene segments, respectively. Variability in TCR gamma polypeptide size and disulfide linkage is determined by the number of copies and the sequence of a repeated segment of the constant region. Thus C gamma 1 and C gamma 2 are used to generate structurally distinct, yet functional, T3-associated receptor complexes on peripheral blood lymphocytes. Tryptic peptide mapping suggests that the T3-associated TCR gamma and delta peptides in the nondisulfide-linked form are distinct.

Extensive junctional diversity of rearranged human T cell receptor delta genes.

The human T cell receptor delta (TCR delta) gene encodes one component of the TCR gamma delta-CD3 complex found on subsets of peripheral blood and thymic T cells. Human TCR delta diversity was estimated by characterizing rearrangements in TCR gamma delta cell lines and determining the structures of complementary DNA clones representing functional and nonfunctional transcripts in these cell lines. One V delta segment and one J delta segment were identified in all functional transcripts, although a distinct J delta segment was identified in a truncated transcript. Further, one D delta element was identified, and evidence for the use of an additional D delta element was obtained. Thus human TCR delta genes appear to use a limited number of germline elements. However, the apparent use of two D delta elements in tandem coupled with imprecise joining and extensive incorporation of N nucleotides generates unprecedented variability in the junctional region.

In-situ straining and time-resolved electron tomography data acquisition in a transmission electron microscope.

This paper reports the preliminary results of a new in-situ three-dimensional (3D) imaging system for observing plastic deformation behavior in a transmission electron microscope (TEM) as a directly relevant development of the recently reported straining-and-tomography holder [Sato K et al. (2015) Development of a novel straining holder for transmission electron microscopy compatible with single tilt-axis electron tomography. Microsc. 64: 369-375]. We designed an integrated system using the holder and newly developed straining and image-acquisition software and then developed an experimental procedure for in-situ straining and time-resolved electron tomography (ET) data acquisition. The software for image acquisition and 3D visualization was developed based on the commercially available ET software TEMographyTM. We achieved time-resolved 3D visualization of nanometer-scale plastic deformation behavior in a Pb-Sn alloy sample, thus demonstrating the capability of this system for potential applications in materials science.

Antiteratogenic effects of tumor inhibitors, caffeine, antipain, and retinoic acid in mice.

To learn the effects of tumor inhibitors on chemically induced malformations, caffeine, antipain, and 13-trans-retinoic acid were given to pregnant ICR/Jcl mice after a single dose of urethan, N-hydroxyurethan, N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea, or 4-nitroquinoline 1-oxide, which induces about 50% of the malformed fetuses. When caffeine was given immediately after carcinogen treatment on Day 10, urethan- and N-ethyl-N-nitrosourea-induced malformations were significantly suppressed by caffeine posttreatment, while N-hydroxyurethan- and N-methyl-N-nitrosourea-induced malformations were not suppressed by caffeine. 4-Nitroquinoline 1-oxide-initiated teratogenesis was also suppressed, but not significantly so (p not equal to 0.07). The results were very similar to those of the effects of caffeine on tumors induced by these carcinogens. Malformations of genetic origin (cleft palates and cleft lips) in CL/Fr mice were also suppressed significantly by caffeine treatment on Days 8 to 11, although the level of inhibition was less than that in chemically induced malformations. A protease inhibitor (antipromotor), antipain, also suppressed urethan-induced malformations. The antiteratogenic effects of antipain were most effective when it was given during the period of 24 to 48 hr after urethan treatment, while those of caffeine were most effective when it was given immediately after urethan. The promoting process might be involved in chemically induced teratogenesis, as it was in carcinogenesis. A natural retinoid (13-trans-retinoic acid) also suppressed urethan-induced malformations. Thus, tumors and malformations induced by chemical carcinogens were suppressed by tumor inhibitors, suggesting the similarity of both processes in the subcellular level, in spite of their morphological differences.

Molecular cloning and functional expression of a cDNA for mouse squalene synthase.

Using a probe obtained by PCR amplification, a full-length cDNA encoding squalene synthase was isolated from a mouse liver cDNA library. Its nucleotide sequence had an open reading frame fro a 416 amino acid polypeptide (calculated molecular mass, 48 kDa). In vitro transcription of the cDNA followed by in vitro translation produced a protein of the expected size. The deduced amino acid sequence was 93%, 88% and 46% identical to those of the rat, human and budding yeast squalene synthases, respectively. Blotting analyses showed that the mRNA is 1.6 kb in size and that less than two copies of the gene are present in the mouse genome. To establish the enzyme activity, the entire coding region was subcloned into an expression plasmid so that it was in frame with the N-terminal region of beta-galactosidase. Escherichia coli, which was transformed with the recombinant plasmid, expressed high activity of converting farnesyl diphosphate into squalene.

A novel isoform of rat hepatocyte nuclear factor 4 (HNF-4).

Based on the published nucleotide sequence for rat hepatocyte nuclear factor 4 (HNF-4; Sladek, F.M., Zhong, W., Lai, E. and Darnell, J.E., Jr. (1990) Genes Dev. 4, 2353-2365), we have cloned a cDNA by means of polymerase chain reaction amplification of reverse-transcribed RNA (RT-PCR). Our clone contained an extra segment of 30 bp, which was not found in the previously reported clone, in the coding region near the C-terminus. Further RT-PCR analysis demonstrated that both isoforms of HNF-4 mRNA, i.e., with or without the 30 nucleotide segment, occur in rat liver and kidney, presumably by differential splicing.

cDNA cloning of a putative G protein-coupled receptor from brain.

Using degenerate oligonucleotide primers corresponding to conserved regions of the G-protein coupled receptor superfamily, we carried out a low-stringency polymerase chain reaction and obtained two novel partial-length clones from a rat brain cDNA library. We used one of these clones for conventional library screening and isolated a longer cDNA clone, designated as RBU-15, from another rat brain library. Although RBU-15 was truncated at its 5' end, Northern blot analysis revealed that the gene was expressed in the brain and spleen. Next, we isolated a full-length cDNA clone, designated as HB-954, from a human fetal brain library, using RBU-15 as a probe. The deduced amino acid sequence of HB-954 contained four putative glycosylation sites in the N-terminal part, seven transmembrane domains, and a large cytosolic domain in the C-terminal part. The protein products of RBU-15 and HB-954 likely belong to a distinctive subfamily, because no receptors in the superfamily were found to be closely related to them.

cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases.

Using degenerate oligonucleotide primers corresponding to conserved regions of the calcium-dependent protein kinase (CDPK) family, we carried out a polymerase chain reaction and obtained four distinct partial-length cDNAs from a maize leaf library. We then used these clones as probes for conventional screening and isolated 19 longer clones from another cDNA library of maize seedlings. These clones were classified into four groups based on their DNA cross-hybridization. Two full-length cDNAs, designated as ZmCDPK9 and ZmCDPK7, were sequenced and characterized. The predicted protein of each clone was a typical CDPK with eleven canonical subdomains of protein kinases, and four EF-hand calcium-binding motifs in its N-terminal and C-terminal halves, respectively. The catalytic and regulatory domains were linked by a well-conserved junction domain. The N-terminus of the protein also contained a consensus sequence for an N-myristoylation signal. Northern blot analysis showed that the transcription level of each gene was higher in roots and etiolated leaves than in green leaves. To confirm the calcium dependency of the maize enzymes, the entire coding region of ZmCDPK9 was subcloned into an expression vector so that it was in frame with the vector-encoded peptide tags. A cell-free extract of Escherichia coli transformed with the recombinant plasmid exhibited calcium-dependent phosphorylation activity, using casein as a substrate.

Nucleotide sequence of a cDNA for mouse squalene epoxidase.

A full-length cDNA encoding mouse squalene epoxidase was isolated by screening a mouse liver cDNA library with the rat squalene epoxidase gene as a probe. The cDNA had an open reading frame for a 572 amino acid polypeptide with a calculated molecular mass of 63.8 kDa. The predicted amino acid sequence of the mouse enzyme contained an FAD-binding motif, and was 93% identical to that of the rat enzyme. The former is one amino acid shorter than the latter. Blotting analyses showed that the mRNA is 2.8 kb in size and that a single copy of the gene is present in the mouse genome.

Identification of two splice isoforms of mRNA for mouse hepatocyte nuclear factor 4 (HNF-4).

Hepatocyte nuclear factor 4 (HNF-4) is a liver-enriched transcription factor involved in the expression of many liver-specific genes. In the preceding communication (Hata, S., Tsukamoto, T. and Osumi, T. (1992) Biochim. Biophys. Acta 1131, 211-213), we reported the presence of two isoforms of mRNA for HNF-4 in rat liver and kidney. The longer isoform contained a segment of 30 bases which was not present in the shorter one. As an initial step to determine whether or not other mammals have these mRNA isoforms, we isolated a cDNA for mouse HNF-4 using the rat HNF-4 gene as a probe. The cDNA had an open reading frame for a 465 amino acid polypeptide. The deduced amino acid sequence was remarkably conserved between mouse HNF-4 and rat HNF-4 (99.6% identical). Moreover, like the cDNA for the larger rat isoform, the mouse cDNA contained an extra segment of 30 bp in the coding region near the C-terminus. Blotting analyses showed that the mRNA is about 3.7 kb in size and that a single copy of the gene is present in the mouse genome. Next we carried out the polymerase chain reaction (PCR) using primers located just upstream and downstream of the extra segment. Two PCR products were amplified from a mouse liver cDNA library. Determination of their nucleotide sequences proved that they exactly corresponded to the two rat isoforms. Finally, we amplified a DNA fragment (1.1 kb in size) from mouse genomic DNA using the same PCR primers as above. Its nucleotide sequence unequivocally confirmed that different splice donor sites were used to generate the two isoforms.

cDNA cloning and expression analysis of a non-photosynthetic ferredoxin gene in morning glory (Pharbitis nil).

A full-length cDNA encoding a non-photosynthetic ferredoxin was isolated from apical buds of morning glory (Pharbitis nil), a short-day plant, by differential screening under flower-inducing and non-inducing conditions. Northern analysis and in situ hybridization showed that the transcript was abundant in shoot apices and root tips. The transcript level in the apical buds decreased with the flower-inducing light treatment.