Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.     

The impact of diagnostic tests in evaluating patients with syncope

We reviewed the charts of 100 patients admitted to the hospital for evaluation of syncope. The charts were examined with special attention given to the causes of syncope, the frequency and benefit of diagnostic tests, and the relative cost of these tests. In 39 patients no etiology for syncope was found, and another 18 were felt to have had a vasovagal episode. Twelve patients had arrhythmias as the cause for syncope. Most of the patients underwent a variety of diagnostic tests including cardiac enzyme determinations, brain scans, electroencephalograms, head CAT scans, and Holter monitoring. In most instances, these tests added little useful information to the initial history and physical exam and were done at great expense to the patient. Our data suggest that extensive neurologic testing in patients with "routine" syncope is not warranted and that the focus of hospitalization should be to rule out potentially life-threatening arrhythmias.     

Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene

A 180,388-bp contig encompassing the human retinoblastoma gene was sequenced in its entirety. Partial analysis of the sequence revealed (1) a high (A + T)/(G + C) ratio and a high density of Line-1 (L1) repeat sequences, suggesting that the locus maps to G-bands 13q14.12 or 13q14.2; (2) Alu repeats that are asymmetrically oriented over a region extending 87 kb; (3) an overabundance of non-Alu-associated poly(A) tracts 10 bp or larger oriented in the antisense rather than the sense direction (36 vs 6); (4) an Alu sequence nested within an L1 repeat, indicating that the expansion of L1 repeats predates at least some of the Alu expansions; (5) at least three newly discovered microsatellite polymorphisms, one of which was subsequently found to be identical to a polymorphism in a microsatellite-based linkage map of the human genome published by another group; and (6) the basis of previously discovered intragenic RFLPs. This sequence should enhance studies of this locus and of the organization of the human genome.     

River ecosystem resilience to extreme flood events

Floods have a major influence in structuring river ecosystems. Considering projected increases in high‐magnitude rainfall events with climate change, major flooding events are expected to increase in many regions of the world. However, there is uncertainty about the effect of different flooding regimes and the importance of flood timing in structuring riverine habitats and their associated biotic communities. In addition, our understanding of community response is hindered by a lack of long‐term datasets to evaluate river ecosystem resilience to flooding. Here we show that in a river ecosystem studied for 30 years, a major winter flood reset the invertebrate community to a community similar to one that existed 15 years earlier. The community had not recovered to the preflood state when recurrent summer flooding 9 years later reset the ecosystem back to an even earlier community. Total macroinvertebrate density was reduced in the winter flood by an order of magnitude more than the summer flood. Meiofaunal invertebrates were more resilient to the flooding than macroinvertebrates, possibly due to their smaller body size facilitating greater access to in‐stream refugia. Pacific pink salmon escapement was markedly affected by the winter flood when eggs were developing in redds, compared to summer flooding, which occurred before the majority of eggs were laid. Our findings inform a proposed conceptual model of three possible responses to flooding by the invertebrate community in terms of switching to different states and effects on resilience to future flooding events. In a changing climate, understanding these responses is important for river managers to mitigate the biological impacts of extreme flooding effects.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Interaction between clonal plasma cells and the immune system in plasma cell dyscrasias

The term "monoclonal gammopathy" (MG) includes a group of clonal plasma cell disorders, which show heterogeneous clinical behavior. While multiple myeloma (MM) and plasma cell leukemia (PCL) are incurable malignant diseases, most patients with MG of undetermined significance (MGUS) show an indolent/benign clinical course. Evidence has accumulated which supports the role of the bone marrow microenvironment in MG. Accordingly, the survival, drug-resistance and proliferation of MM cells have been shown to be largely dependent on a supportive microenvironment. Among the different environment-associated parameters, those related to the status/activity of the immune system are particularly relevant. This review focuses on the different ways clonal plasma cells (PC) interact with the immune system in different models of MG, to characterize crucial events in the development and progression of MG. These advances may support the design of novel therapeutic approaches in patients with MG.     

Quantitative detection of methylated SOCS-1, a tumor suppressor gene, by a modified protocol of quantitative real time methylation-specific PCR using SYBR green and its use in early gastric cancer detection

Although methylation-specific PCR (MSP) is a sensitive technique in the detection of DNA hypermethylation, it is not quantitative. Here we described a modified PCR protocol to quantify methylated SOCS-1 gene by real time MSP using SYBR green, which involves an additional PCR step after the 72 degrees C extension step. This modified protocol is also useful in the quantitative detection of methylated SOCS-1 gene in serum samples of gastric cancer patients.