Assessing venous thrombus in renal cell carcinoma: preliminary results for unenhanced 3D-SSFP MRI.

AIM: To test the potential of unenhanced cardiac- and respiratory-motion-corrected three-dimensional steady-state free precession (3D-SSFP) magnetic resonance imaging (MRI) for the assessment of inferior vena cava (IVC) thrombus in patients with clear-cell renal cell carcinoma (cRCC), compared to standard contrast-enhanced (CE)-MRI and CE-computed tomography (CT). MATERIALS AND METHODS: Eighteen patients with cRCC and IVC thrombus, who received CE-MRI and 3D-SSFP at 1.5 T between June 2015 and December 2017, were included. The diagnostic performance of 3D-SSFP in determining the level of thrombus extension, contrast-to-noise ratio (CNR), and image quality were compared with standard MRI/CT and validated against intraoperative and histopathology results. RESULTS: There was 100% agreement between 3D-SSFP, 83.3% agreement between CE-MRI, and 71.4% agreement between CE-CT and surgical findings regarding the level of IVC thrombus. In addition, 3D-SSFP showed a slightly superior estimate of pathological IVC volume. 3D-SSFP reached a significantly higher CNR in the supra- and infrarenal IVC compared to the morphological sequence T2-weighted half-Fourier axial single-shot fast spin-echo (T2-HASTE) and all phases of CE-MRI. More specifically, 3D-SSFP showed a significantly higher CNR in the infrarenal IVC (mean CNR of 10.09±5.74 versus 4.21±2.33 in the delayed phase, p≤0.001) and in the suprarenal IVC (mean CNR of 9.22±4.11 versus 4.84±5.74 in the late arterial phase, p=0.015). CE-CT also was significantly inferior to 3D-SSFP (p≤0.01) and slightly inferior to CE-MRI (p>0.05). The thrombus delineation score for 3D-SSFP (4.38±0.67) was higher compared to CE-MRI (3.76±0.56, p=0.005). CONCLUSION: This preliminary study indicates that 3D-SSFP can achieve an accurate assessment of IVC thrombus in cRCC patients without the need for contrast medium administration, being superior to standard MRI and CT.

Towards characterization of photo-excited electron transfer and catalysis in natural and artificial systems using XFELs.

The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ray emission spectra, using an energy dispersive spectrometer, at ambient conditions, and used this approach to study the room temperature structure and intermediate states of the photosynthetic water oxidizing metallo-protein, photosystem II. Moreover, we have also used this setup to simultaneously collect the X-ray emission spectra from multiple metals to follow the ultrafast dynamics of light-induced charge transfer between multiple metal sites. A Mn-Ti containing system was studied at an XFEL to demonstrate the efficacy and potential of this method.

Simulations of the two-dimensional electronic spectroscopy of the photosystem II reaction center.

We report simulations of the two-dimensional electronic spectroscopy of the Q(y) band of the D1-D2-Cyt b559 photosystem II reaction center at 77 K. We base the simulations on an existing Hamiltonian that was derived by simultaneous fitting to a wide range of linear spectroscopic measurements and described within modified Redfield theory. The model obtains reasonable agreement with most aspects of the two-dimensional spectra, including the overall peak shapes and excited state absorption features. It does not reproduce the rapid equilibration from high energy to low energy excitonic states evident by a strong cross-peak below the diagonal. We explore modifications to the model to incorporate new structural data and improve agreement with the two-dimensional spectra. We find that strengthening the system-bath coupling and lowering the degree of disorder significantly improves agreement with the cross-peak feature, while lessening agreement with the relative diagonal/antidiagonal width of the 2D spectra. We conclude that two-dimensional electronic spectroscopy provides a sensitive test of excitonic models of the photosystem II reaction center and discuss avenues for further refinement of such models.

Phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in North America and the UK.

Genetic relationships between 35 spring viremia of carp virus (SVCV) genogroup Ia isolates were determined based on the nucleotide sequences of the phosphoprotein (P) gene and glycoprotein (G) genes. Phylogenetic analysis based on P gene sequences revealed 2 distinct subgroups within SVCV genogroup Ia, designated SVCV Iai and Iaii, and suggests at least 2 independent introductions of the virus into the USA in 2002. Combined P- and G-sequence data support the emergence of SVCV in Illinois, USA, and in Lake Ontario, Canada, from the initial outbreak in Wisconsin, USA, and demonstrate a close genetic link to viruses isolated during routine import checks on fish brought into the UK from Asia. The data also showed a genetic link between SVCV isolations made in Missouri and Washington, USA, in 2004 and the earlier isolation made in North Carolina, USA, in 2002. However, based on the close relationship to a 2004 UK isolate, the data suggest than the Washington isolate represents a third introduction into the US from a common source, rather than a reemergence from the 2002 isolate. There was strong phylogenetic support for an Asian origin for 9 of 16 UK viruses isolated either from imported fish, or shown to have been in direct contact with fish imported from Asia. In one case, there was 100% nucleotide identity in the G-gene with a virus isolated in China.

Characterization and mutational studies of equine infectious anemia virus dUTPase.

The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages. Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis. Specific activities of preparations ranged from 4 x 10(3) to 5 x 10(4) units/mg. Recombinant EIAV dUTPase was highly specific for dUTP with a Km in the range of 3 to 8 microM. The enzyme was sensitive to inhibition by dUDP with little inhibition by other nucleotides or the reaction products, dUMP and PPi. The subunit organization of recombinant EIAV dUTPase was probed by gel filtration, glycerol gradient centrifugation, and chemical cross-linking, and is a trimer. We have begun mutational analyses by targeting a conserved domain present at the carboxyl terminus of all dUTPases that shares high homology to the phosphate binding loops (P-loops) of a number of ATP- and GTP-binding phosphatases. The P-loop-like motif of dUTPases is glycine rich but lacks the invariant lysine found in authentic P-loops. Deletion of this motif leads to loss of dUTPase activity; a series of point mutations that have been shown to inactivate authentic P-loops also abolish EIAV dUTPase activity.

Calcium-sensing receptor expression and regulation by extracellular calcium in the AtT-20 pituitary cell line.

A 120 kDa, G protein-coupled calcium-sensing receptor (CaR) was recently identified and cloned from bovine parathyroid and rat kidney. We report here that a similar calcium-sensing receptor is also present in rat and mouse pituitary as well as in the mouse pituitary cell line, AtT-20. Fragments (383-bp) of the extracellular domain of the calcium-sensing receptor from the AtT-20 cells and mouse pituitary were amplified by RT-PCR, sequenced, and found to be identical. By Northern blot analysis, AtT-20 cells expressed a major CaR mRNA transcript of 7.5 kb and three minor transcripts of 9.5, 4.0, and 1.5 kb. Except for the 9.5-kb species, these CaR transcripts were also found to be present in mouse kidney, where the 7.5-kb transcript was again the predominant form. The presence of the CaR protein in AtT-20 cells was documented directly by fluorescence immunocytochemistry using an antibody directed against the extracellular domain of the CaR. Exposure of AtT-20 cells to increasing extracellular calcium concentrations from 0.3 t 3 mM for 24 h resulted in a 2- to 4-fold increase in the levels of CaR mRNA, but not of the RNAs for beta-actin or POMC. The CaR appeared to be functional in AtT-20 cells, since acute increases in extracellular calcium between 2 and 5 mM induced increases in the cellular content of total inositol phosphates, cytosolic calcium, and cAMP. This report suggests that pituitary cells respond to changes in extracellular calcium via a G protein-coupled CaR.

Duration of action of hypertonic saline on mucociliary clearance in the normal lung.

Inhalation of hypertonic saline (HS) acutely enhances mucociliary clearance (MC) in both health and disease. In patients with cystic fibrosis (CF), repeated use of HS causes a sustained improvement in MC as well as clinical benefit. The pharmacodynamic duration of activity on MC may be an important determinant of its therapeutic potential in other airways diseases. Before moving toward testing the clinical benefits of HS for non-CF indications, we sought to assess the duration of pharmacodynamic effects of HS in healthy subjects by performing radiotracer clearance studies at baseline, 30-min post-HS administration, and 4-h post-HS administration. Indeed, acceleration of MC was observed when measured 30 min after HS inhalation. This acceleration was most pronounced in the first 30 min after inhaling the radiotracer in the central lung region (mean Ave30Clr = 15.5 vs. 8.6% for 30-min post-HS treatment vs. mean baseline, respectively, P < 0.005), suggesting that acute HS effects were greatest in the larger bronchial airways. In contrast, when MC was measured 4 h after HS administration, all indices of central lung region MC were slower than at baseline: Ave30Clr = 5.9% vs. 8.6% (P = 0.10); Ave90Clr = 12.4% vs. 16.8% (P < 0.05); clearance through 3 h = 29.4 vs. 43.7% (P < 0.002); and clearance through 6 h = 39.4 vs. 50.2% (P < 0.02). This apparent slowing of MC in healthy subjects 4-h post-HS administration may reflect depletion of airway mucus following acute HS administration.

The reduced responsiveness of cultured bovine parathyroid cells to extracellular Ca2+ is associated with marked reduction in the expression of extracellular Ca(2+)-sensing receptor messenger ribonucleic acid and protein.

PTH secretion from dispersed bovine parathyroid cells maintained in culture becomes progressively less responsive to changes in the extracellular Ca2+ concentration (Ca2+o) over several days. We have now investigated whether this change in secretory control is associated with alterations in the expression of the Ca2+o-sensing receptor (BoPCaR) recently cloned from bovine parathyroid, which plays a central role in Ca2+o-regulated PTH secretion. BoPCaR messenger RNA levels dropped rapidly in cultured bovine parathyroid cells, as assessed by Northern analysis, decreasing by 78% within 18 h and remaining low for at least 4 days. The level of receptor protein decreased to a comparable extent (approximately 72-82%) after 3-4 days in culture, as determined by immunocytochemistry with specific antibodies directed at the extracellular domain of the receptor. The half-time for the reduction in receptor protein (approximately 2 days) was considerably longer, however, than that for BoPCaR messenger RNA, but was comparable to that for the loss of sensitivity of PTH secretion to Ca2+o. Indeed, there was a close linear correlation between maximal suppressibility of PTH secretion and the intensity of staining for the receptor protein (r = 0.88; P = 0.004). We conclude that alterations in the expression of BoPCaR could explain much of the reduced responsiveness of cultured bovine parathyroid cells to Ca2+o.

A family of cloning vectors containing the lacUV5 promoter.

A family of plasmids containing short pieces of Escherichia coli lac promoter DNA has been constructed. DNA fragments from any source may be inserted directly into the unique EcoRI sites of some of these plasmids to achieve transcription under the control of the lacUV5 promoter. Alternatively, the plasmids serve as convenient sources of lac DNA fragments ('portable promoters') containing the 'up' promoter mutations UV5 or Ps (super promoter) as well as the wild-type promoter. pOP95-2, pOP95-5, pOP203-1, pOP203-2 and pOP203-3 are derivatives of pMB9 while pOP95-15 and pOP203-13 are derivatives of pBR322. The pOP95 plasmids contain the 95-bp AluI lac fragment. This fragment includes the UV5 promoter (minus the CAP binding site), the repressor binding site, and ends 2 bp before an ATG encoding the beta-Gal start codon. The pOP203 plasmids contain the 203-bp HaeIII lac fragment. This fragment contains the UV5 promoter (including the L8 mutation in the CAP binding site), the repressor binding site and sequences encoding the first 8 amino acids of beta-Gal. To shorten and introduce reading frame heterogeneity in the beta-Gal coding end of the pOP203 plasmids, the EcoRI site in pOP203-12 was moved upstream by digesting EcoRI cut plasmid DNA with T4 DNA polymerase and S1 nuclease followed by ligation in the presence of EcoRI linker. This produced the plasmids pOP203-24, pOP203-27, pOP203-28 and pOP203-29. pOP203-29 encodes essentially just that portion of the beta-Gal mRNA sequence which is protected from nuclease digestion by the bound ribosomal complex (Maizels, 1974).

Sequence and structural organization of the human gene encoding ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a potent polypeptide hormone whose actions appear to be restricted to the nervous system where it promotes survival, neurotransmitter synthesis and neurite outgrowth in certain neuronal populations. We have cloned the gene encoding human CNTF (hCNTF) and have characterized its structure and organization. The hCNTF gene appears to be a unique-copy gene with a simple genetic organization, since only a single intron interrupts the coding domain. The hCNTF gene is located on chromosome 11, as determined using human-hamster somatic cell hybrids. The CNTF protein is highly conserved in evolution. The amino acid (aa) sequences of rat and rabbit CNTF translated from cDNAs display approx. 85% homology with the deduced aa sequence encoding hCNTF.

Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3' untranslated region identifies the virus as a close relative of infectious bronchitis virus.

The 3' end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3' untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3' UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3' UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3' UTRs provide additional evidence that these avian coronaviruses are closely related.

Ca2+ receptor mRNA and protein increase in the rat parathyroid gland with advancing age.

Parathyroid hormone (PTH) release is regulated by extracellular calcium through a Ca2+ receptor (CaR) located on the surface of the parathyroid cell. With advancing age, the serum concentration of PTH increases, and evidence suggests that the calcium set-point for PTH release may also increase. To determine whether these changes are linked to a change in CaR expression, we quantitated mRNA and protein for the receptor in parathyroid glands of 6-week-, 6-month- and 24-month-old rats. Thyroid and kidney tissue were also studied. Between 6 weeks and 24 months of age, CaR mRNA in the parathyroid gland increased 11.4- and 3.3-fold as measured by competitive reverse transcription PCR and solution hybridization assays respectively. Message levels for the receptor also increased in the thyroid but not in the kidney. Coincident with the increase in message levels, receptor protein concentration in the parathyroid increased 7-fold between 6 weeks and 24 months of age. These results suggest that the altered relationship between extracellular calcium and PTH release observed in aging is associated with dramatic changes in CaR metabolism. That PTH secretion is increased despite increased receptor concentration suggests that aging may impair calcium binding or coupling between the CaR and down-stream effector elements in the pathway regulating PTH release.

Effects of testosterone on protein synthesis in rat seminal vesicles analysed by two-dimensional gel electrophoresis.

Proteins synthesised by seminal vesicles of normal rats were labelled with [35S]methionine and were then separated using two-dimensional gel electrophoresis. Isoelectric focusing or nonequilibrium pH gradient electrophoresis were used in the first dimension followed by polyacrylamide gel electrophoresis under denaturing conditions. Using antiserum to total seminal vesicle secretion and running the immuno-precipitated proteins on two-dimensional gels, secretory proteins were identified and shown to be much more complex than previously thought. Proteins synthesised by seminal vesicles from rats castrated 1-2 weeks before were also labelled with [35S]methionine and separated on two-dimensional gels. Comparison of the two-dimensional protein maps from normal and castrated animals showed that a substantial number of proteins were differentially induced or repressed by testosterone. Of the secretory proteins, some were clearly regulated in a highly differential manner but others appeared to be unaffected by castration. The results are discussed in relation to previous measurements of mRNA sequence complexity and show that previous conclusions derived from using less sophisticated methods are oversimplifications of the response.

Synthesis of androgen-dependent secretory proteins by rat seminal vesicles.

Androgenic steroids control the synthesis and secretion of several proteins by the seminal vesicles of the male rat. Prominent among them are 2 basic proteins, S and F, previously used as markers of androgen action. These proteins are not found among translation products of a wheat-germ protein-synthesising system supplied with mRNA from seminal vesicles of normal rats. Instead, higher molecular weight forms, S' and F', are formed which are nonetheless related antigenically to S and F respectively. Injected into Xenopus Laevis oocytes, seminal vesicle mRNA does direct synthesis and secretion of polypeptides S and F. Partial peptide analysis confirms that S' and F' have extensive amino acid sequence homology with S and F respectively. We conclude that S' and F' are precursor forms of the secreted proteins and thus at least 2 abundant proteins of this male accessory tissue are secreted by a mechanism common to a wide number of secreted proteins.

Isolation of cells from rat seminal vesicles and epididymis and their use in studying androgen action.

Cells isolated enzymically from seminal vesicles and epididymides of normal and castrated rats were shown by electron microscopy to be intact and representative of the tissue. The cells synthesize and secrete tissue-specific proteins. Short-term incorporation of [3H]uridine and [35S]methionine was measured to determine the effects of castration on RNA and protein synthesis. Epididymal cells and tissue incorporated uridine at similar rates which were unaltered by castration. Similarly castration failed to diminish uridine incorporation by seminal vesicle cells and tissue. Therefore, androgens may principally control RNA degradation. A similar situation pertained to methionine incorporation by epididymal cells and tissue so here too control may be via protein degradation. In contrast, castration greatly decreased methionine incorporation by seminal vesicle tissue but not by isolated cells. Isolated cells were more active than in tissue, particularly those from castrated rats, and may be released from stromal-epithelial interactions and controls.

Malignant disseminated porokeratosis.

A patient has been observed with a distinct form of disseminated porokeratosis. During the course of his disease, he developed changes in the porokeratosis lesions that included cellular atypia, dysplasia, and invasive squamous cell carcinoma. One of the squamous cell carcinomas produced regional and disseminated metastases, resulting in the death of the patient. Although malignancy has been previously described in the various types of porokeratosis, this is the first report of disseminated metastases and death in any form of this disease.

Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10.

Four lambs and 3 calves, seronegative to bluetongue virus (BTV), were inoculated intravenously with a highly plaque-purified strain of BTV Serotype 10. A single calf and lamb served as controls and were inoculated with uninfected cell culture lysate. All BTV-inoculated lambs exhibited mild clinical manifestations of bluetongue, whereas infected calves were asymptomatic. Viremia persisted in BTV-infected lambs for 35-42 days, and for 42-56 days in BTV-infected calves. Neutralizing antibodies were first detected in sera collected at Day 14 post-inoculation (PI) from 2 BTV-infected calves and all 4 infected lambs, and at Day 28 PI in the remaining calf. The appearance of neutralizing antibody in serum did not coincide with clearance of virus from blood; BTV and specific neutralizing antibody coexisted in peripheral blood of infected lambs and calves for as long as 28 days. The sequential development, specificity and intensity of virus protein-specific humoral immune responses of lambs and calves were evaluated by immunoprecipitation of [35S]-labelled proteins in BTV-infected cell lysates by sera collected from inoculated animals at bi-weekly intervals PI. Sera from infected lambs and calves reacted most consistently with BTV structural proteins VP2 and VP7, and nonstructural protein NS2, and less consistently with structural protein VP5, and nonstructural protein NS1. Lambs developed humoral immune responses to individual BTV proteins more rapidly than calves, and one calf had especially weak virus protein-specific humoral immune responses; viremia persisted longer in this calf than any other animal in the study. The clearance of virus from the peripheral blood of BTV-infected lambs and calves is not caused simply by the production of virus-specific neutralizing antibody, however the intensity of humoral immune responses to individual BTV proteins might influence the duration of viremia in different animals.

Phorbol ester stimulation of equine macrophage cultures alters expression of equine infectious anemia virus.

Equine infectious anemia virus (EIAV) is a lentivirus that replicates predominantly in mature tissue macrophages. Viral expression is strongly influenced by the state of differentiation of the host cell. While blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. Activation of mature macrophages infected with EIAV might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid sequences within the long terminal repeat (LTR). Using DNA amplification techniques, we compared LTR sequences of U.S. field strains of EIAV to sequences of a laboratory adapted strain of the virus. All field strain sequences were more closely related to Wyoming strain than to the Malmquist laboratory adapted strain or a previously sequenced infectious molecular clone of EIAV. Primary equine monocyte-derived macrophage cultures were infected with virulent and avirulent strains of EIAV and the effects of macrophage stimulation on EIAV expression were determined. Stimulation of macrophages with phorbol ester activated the cells to secrete tumor necrosis factor alpha (TNF alpha). This activation signal also resulted in a significant downregulation of viral expression as determined by supernatant reverse transcriptase activity. This effect occurred independent of the virulence of the virus strain used or the nucleic acid sequence of the viral LTR. This may represent an adaptive response of EIAV to evade the host immune response and establish a persistent infection.

Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes.

An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of lipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins (150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes.

Turkey coryza: lack of correlation between plasmids and pathogenicity of Bordetella avium.

Plasmids were removed from pathogenic Bordetella avium using a variety of treatments. The plasmid-cure rates depended on the treatment and isolate. Pathogenicity of B. avium in turkey poults was not altered by removal of plasmids.