Fluorophotometric enzyme immunoassay of thyroid-stimulating hormone.

A method for enzyme immunoassay of thyroid-stimulating hormone (TSH) is described, TSH was conjugated with horseradish peroxidase according to periodate oxidation method. Separation of the bound and free was obtained by double-antibody solid-phase technique using Sepharose 4B-anti-rabbit immunogiobulin G (IgG)-geat IgG. The fluorescence reaction using tyramine and hydrogen peroxide as substrates was used for the determination of enzyme activity in order to increase the sensitivity of enzyme immunoassay. The standard curve for serum TSH was satisfactory to recognize TSH concentrations as 0.06 μU/tube. TSH values obtained by this method correlated well with those obtained by radioimmunoassay (r, 0.96). The coefficients of variation were 1.8 to 5.3% (within assay) and 5.1 to 10.5% (between assay). The method is about equal to radioimmunoassay with respect to sensitivity. Since it requires minimal equipment and is less expensive than radioimmunoassay, it is possible to perform routine assays even in laboratories with limited facilities.     

The Gene Encoding Flavanone 3-Hydroxylase is Expressed Normally in the Pale Yellow Flowers of the Japanese Morning Glory Carrying the speckled Mutation Which Produce Neither Flavonol nor Anthocyanin but Accumulate Chalcone, Aurone and Flavanone

The Japanese morning glory carrying the recessive mutable speckledallele with the dominant speckled-activator bears colorlessflowers with fine and round colored spots distributed over thecorolla whereas the plant without the speckled-activator producespale yellow flowers. Previous chemical analysis has indicatedthat a mutation in the gene for flavanone 3-hydroxylase (F3H)is a likely candidate for the speckled allele. However, theF3HmRNA without sequence alteration accumulates normally inthe pale yellow flowers, indicating that the speckled alleleis neither the F3H gene nor a regulatory gene acting on theF3H gene expression. (Received April 4, 1997; Accepted June 2, 1997)     

Evolution of green lacewings (Neuroptera: Chrysopidae): a molecular supermatrix approach

We present a time‐calibrated phylogeny of the charismatic green lacewings (Neuroptera: Chrysopidae). Previous phylogenetic studies on the family using DNA sequences have suffered from sparse taxon sampling and/or limited amounts of data. Here we combine all available previously published DNA sequence data and add to it new DNA sequences generated for this study. We analysed these data in a supermatrix using Bayesian and maximum likelihood methods and provide a phylogenetic hypothesis for the family that recovers strong support for the monophyly of all subfamilies and resolves relationships among a large proportion of chrysopine genera. Chrysopinae tribes Leucochrysini and Belonopterygini were recovered as monophyletic sister clades, while the species‐rich tribe Chrysopini was rendered paraphyletic by Ankylopterygini. Relationships among the subfamilies were resolved, although with relatively low statistical support, and the topology varied based on the method of analysis. Greatest support was found for Apochrysinae as sister to Nothochrysinae and Chrysopinae, which is in contrast to traditional concepts that place Nothochrysinae as sister to the rest of the family. Divergence estimates suggest that the stem groups to the various subfamilies diverged during the Triassic‐Jurassic, and that stem groups of the chrysopine tribes diverged during the Cretaceous.     

Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determinants of Slow Walking Speed in Ambulatory Patients Undergoing Maintenance Hemodialysis

Walking ability is significantly lower in hemodialysis patients compared to healthy people. Decreased walking ability characterized by slow walking speed is associated with adverse clinical events, but determinants of decreased walking speed in hemodialysis patients are unknown. The purpose of this study was to identify factors associated with slow walking speed in ambulatory hemodialysis patients. Subjects were 122 outpatients (64 men, 58 women; mean age, 68 years) undergoing hemodialysis. Clinical characteristics including comorbidities, motor function (strength, flexibility, and balance), and maximum walking speed (MWS) were measured and compared across sex-specific tertiles of MWS. Univariate and multivariate logistic regression analyses were performed to examine whether clinical characteristics and motor function could discriminate between the lowest, middle, and highest tertiles of MWS. Significant and common factors that discriminated the lowest and highest tertiles of MWS from other categories were presence of cardiac disease (lowest: odds ratio [OR] = 3.33, 95% confidence interval [CI] = 1.26–8.83, P<0.05; highest: OR = 2.84, 95% CI = 1.18–6.84, P<0.05), leg strength (OR = 0.62, 95% CI = 0.40–0.95, P<0.05; OR = 0.57, 95% CI = 0.39–0.82, P<0.01), and standing balance (OR = 0.76, 95% CI = 0.63–0.92, P<0.01; OR = 0.81, 95% CI = 0.68–0.97, P<0.05). History of fracture (OR = 3.35, 95% CI = 1.08–10.38; P<0.05) was a significant factor only in the lowest tertile. Cardiac disease, history of fracture, decreased leg strength, and poor standing balance were independently associated with slow walking speed in ambulatory hemodialysis patients. These findings provide useful data for planning effective therapeutic regimens to prevent decreases in walking ability in ambulatory hemodialysis patients.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Electrophoretic characterization of ribosomal subunits and proteins in apoptosis: specific downregulation of S11 in staurosporine-treated human breast carcinoma cells.

Stimulation of death receptors (Fas on human T-cell leukemia Jurkat cells and tumor necrosis factor receptor-1 on human monoblastic leukemia U937 cells) triggers the specific degradation of 28S ribosomal RNA, and this process may contribute to cell death through the inhibition of protein synthesis. We have developed an analytical method using a polyacrylamide-agarose composite gel to evaluate ribosomal subunits in apoptotic cells (human breast carcinoma MCF-7 cells treated with staurosporine and human 293T cells irradiated with ultraviolet light were used in addition to the two apoptosis systems described above). No alterations were detected by this method, suggesting that apoptosis, including the process of ribosomal RNA degradation, does not cause fragmentation or extensive conformational changes in the ribosome. We also examined the status of 21 different ribosomal proteins in apoptotic cells by immunoblotting with polyclonal antibodies. S11 was specifically downregulated in apoptotic MCF-7 cells and in other apoptotic breast carcinoma cells. Previous studies have shown that S11 is heterogeneously expressed in cancer cells. Taken together, it appears that particular intracellular environments regulate the expression of S11 protein. However, the mechanism by which this process is modulated is as yet unknown. Furthermore, we have demonstrated that our composite gel electrophoresis system can efficiently detect ubiquitination of ribosomal subunits.