SDS agarose gels for analysis of proteins

A new agarose-based protein electrophoresis gel system is described. The system consists of a highly resolving agarose, MetaPhor XR (FMC BioProducts, Rockland, ME, USA) dissolved in urea and TBE buffer and a stacking gel composed of a high gel-strength agarose, SeaKem Gold (FMC BioProducts). TBE containing sodium dodecyl sulfate (SDS) is used as electrophoresis buffer. The disadvantages of traditional agarose gels have been overcome, and several advantages over polyacrylamide gels have been demonstrated. The system is capable of high-resolution separation of small proteins and has a dynamic separation range equivalent to a 4%-20% gradient polyacrylamide gel. Furthermore, the staining of protein bands by Coomassie Brilliant Blue is very uniform in this gel, and depending on the protein, higher detection sensitivity can be obtained compared to SDS polyacrylamide gels. In Western blotting, proteins are more efficiently transferred to the membrane from the agarose gel than from polyacrylamide gels. Finally, the exceptional stability of agarose allows for gels to be precast and stored for a year.     

Skeletal evidence for child abuse: a physical anthropological perspective

Analysis of the skeletal remains of abused children can prove challenging for forensic pathologists and radiographers who are inexperienced in the direct examination of bones. In such cases, radiographically invisible skeletal lesions that document a history of trauma can often be identified by a physical anthropologist with appropriate osteological experience. This is illustrated by cases in which skeletal remains of four murdered children and a mentally handicapped adult produced evidence of antemortem trauma and perimortem injuries that was critical in developing murder cases against the assailants. In these cases, well-healed areas of subperiosteal new bone formation were identified that were below the threshold of radiographic detection. Such injuries provide strong evidence for a history of physical abuse.     

RNA components of Escherichia coli degradosome: evidence for rRNA decay

Recently, we found that a multicomponent ribonucleolytic degradosome complex formed around RNase E, a key mRNA-degrading and 9S RNA-processing enzyme, contains RNA in addition to its protein components. Herein we show that the RNA found in the degradosome consists primarily of rRNA fragments that have a range of distinctive sizes. We further show that rRNA degradation is carried out in the degradosome by RNase E cleavage of A+U-rich single-stranded regions of mature 16S and 23S rRNAs. The 5S rRNA, which is known to be generated by RNase E processing of the 9S precursor, was also identified in the degradosome, but tRNAs, which are not cleaved by RNase E in vitro, were absent. Our results, which provide evidence that decay of mature rRNAs occurs in growing Escherichia coli cells in the RNA degradosome, implicate RNase E in degradosome-mediated decay.     

Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA

The requirement of PTB, polypyrimidine tract binding protein, for internal initiation of translation has been tested using an RNA affinity column to deplete rabbit reticulocyte lysates of PTB. The affinity column was prepared by coupling CNBr-activated Sepharose with the segment of the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA previously shown to bind PTB. Lysates passed through this column were devoid of PTB, and were incapable of internal initiation of translation dependent on the EMCV 5'-untranslated region, while retaining the capacity for translation dependent on ribosome scanning. Full activity for internal initiation was restored by the addition of recombinant PTB at the physiologically relevant concentration of about 5 micrograms/mL. When various PTB deletion mutants were tested, it was found that this activity required virtually the full-length protein. Thus, PTB is an essential protein for internal initiation promoted by the EMCV 5'-untranslated region. However, the PTB-depleted lysate retained the capacity for internal initiation promoted by the 5'-untranslated regions of another cardiovirus, Theiler's murine encephalomyelitis virus, and of the unrelated hepatitis C virus, and in neither case did addition of recombinant PTB stimulate internal initiation. Therefore, PTB is not a universal internal initiation factor that is indispensable in every case of internal ribosome entry.     

Direct immunocytochemical evidence for the transfer of glutamine from glial cells to neurons: use of specific antibodies directed against the d-stereoisomers of glutamate and glutamine

We have raised antibodies against D-stereoisomers of the amino acids glutamate and glutamine. These stereoisomers are not naturally occurring in mammals but can be taken up into cells by transporters that normally handle the endogenous L-amino acids. Exposure of isolated rabbit retinae to 50 microM D-glutamate resulted in a strong accumulation of D-glutamate, and hence immunoreactivity for D-glutamate in radial glial cells (Müller cells). By contrast the glutamatergic ganglion cells exhibited no immunoreactivity for D-glutamate. D-Glutamate can be converted into D-glutamine by the glial enzyme glutamine synthetase. Immunolabelling for D-glutamine revealed the presence of D-glutamine in somata of subsets of neurons including the glutamatergic ganglion cells. Labelling was also present in the inner plexiform layer, possibly indicating labelling of neuronal processes. These data indicate that after D-glutamate has been taken up into glial cells it is converted into D-glutamine. This D-glutamine is then exported from the glial cells and taken up by a subset of neurons, including the glutamatergic ganglion cells.     

Direct and indirect effects of chronic physical conditions on depression: a preliminary investigation

We have previously demonstrated that interleukin-2 (IL-2) receptors, IL-2 protein, and mRNA for IL-2 are present in human carcinomas in vitro and in vivo. Carcinoma cells synchronized in the G2/M-phase of the cell cycle express significantly more intracytoplasmic IL-2 as well as IL-2R-beta and -gamma than tumor cells in the G0/G1-phase. Here we evaluated immunohistologically the cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen and expression of the cytokine IL-2 in four different carcinoma cell lines. In addition, 34 tissue samples from patients with squamous cell carcinomas of the head and neck were simultaneously analyzed for Ki-67 and IL-2 expression and the data were correlated to the histological grade of the tumors. All tumor cell lines were shown to express IL-2 in the Golgi complex. The strongest IL-2 expression was seen in tumor cells undergoing mitosis, identified by double staining with the antibody to Ki-67. In the tumor tissue, the highest level of co-expression of IL-2 and Ki-67 was observed in poorly differentiated carcinomas, with a labeling index (LI) of 67. 2% for IL-2 and 68.8% for Ki-67. Well-differentiated carcinomas showed a significantly lower expression of both proteins (LI 35.0% for IL-2 and 26.5% for Ki-67). The correlation between the labeling indices was statistically significant (r = 0.747; p<0.001). These results demonstrate that IL-2 expression in human carcinoma tissues is strongly associated with cell proliferation and significantly correlates with the histological tumor grade.     

NMR solution structure of the lead-dependent ribozyme: evidence for dynamics in RNA catalysis

The function of social calls emitted by foraging bats has received little study. Here we use observations of free-ranging greater spear-nosed bats, Phyllostomus hastatus, and field playbacks to determine whether audible, broad-band 'screech' calls attract mates, warn conspecifics or influence access to food. Five lines of evidence suggest that screech calls enable adult females from the same roosting group to fly together from the day roost to feeding sites. (1) Seasonal differences in diet influenced the rate of screech calling recorded outside the cave roost, as well as how often bats departed together. Bats called more often and flew in larger groups when feeding on a concentrated resource, balsa, Ochroma lagopus, flowers, in winter than on more dispersed Cecropia peltata fruit in spring. (2) Observations of bats flying outside the cave, in flyways and at feeding sites indicated that screech calls occurred more often when bats flew in groups than alone. (3) Females from the same roosting group were netted at the same feeding site, sometimes simultaneously, several kilometres from the cave. (4) Calling colour-marked adult females outside the cave were joined by a female group member, both on initial departures and on second foraging trips, more often than non-calling bats. (5) Playbacks attracted conspecifics at roost and feeding sites. Screech calls appear to function as contact calls that recruit and coordinate foraging among group members. We postulate that females benefit from foraging with unrelated roost-mates because they can defend feeding sites more effectively. Copyright 1998 The Association for the Study of Animal Behaviour.     

tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence

We describe a program, tRNAscan-SE, which identifies 99-100% of transfer RNA genes in DNA sequence while giving less than one false positive per 15 gigabases. Two previously described tRNA detection programs are used as fast, first-pass prefilters to identify candidate tRNAs, which are then analyzed by a highly selective tRNA covariance model. This work represents a practical application of RNA covariance models, which are general, probabilistic secondary structure profiles based on stochastic context-free grammars. tRNAscan-SE searches at approximately 30 000 bp/s. Additional extensions to tRNAscan-SE detect unusual tRNA homologues such as selenocysteine tRNAs, tRNA-derived repetitive elements and tRNA pseudogenes.     

The transfer effect in artificial grammar learning: Reappraising the evidence on the transfer of sequential dependencies.

Exposure to sequences of elements generated by an artificial grammar enables observers to classify new sequences presented either with the same (within-domain) or different (across-domain) vocabulary elements as being well- or ill-formed according to that grammar (A. S. Reber, 1969). Experiment 1 replicated G. T. M. Altmann, Z. Dienes, and A. Goode's (1995) demonstration of this effect, but inspection of hits and correct rejections revealed that a single cue was used to reject a subset of the ungrammatical sequences. Experiment 2 removed this cue, and participants no longer discriminated between grammatical and ungrammatical sequences in the novel domain. The authors conclude that G. T. M. Altmann et al.'s demonstration of discrimination in the novel domain did not necessitate the application of knowledge pertaining to sequential dependencies. Implications of the data for other studies, showing abstraction of knowledge across test sequences, are also considered. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

An exploratory investigation into self-other differentiation: Empirical evidence for a monistic perspective on empathy.

Several theoretical definitions of empathy have noted a component of emotional self–other differentiation (ESOD) between the experiences of the therapist and the client. In the present study, an instrument designed to measure the maintenance of emotional separation (MES) was developed. 131 graduate students in social work completed the MES scale, items from the Marlowe-Crowne Social Desirability Scale, and a measure of empathic tendency. Results show a negative relationship between empathy and MES. Findings question the notion that ESOD is a necessary component of empathy and support the idea that ESOD is lost after a certain degree of empathy. Findings also support the author's (1981) conceptualization of empathy as a monistic therapist–client experience. (22 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Direct evidence for suppression of recombination within two pericentric inversions in humans: a new sperm-FISH technique

The tuberculin reaction following the intradermal injection of PPD appears 48-72 hours after injection. The positivity is shown by an > 5 mm area of induration of the skin. Tuberculin reaction is an invaluable instrument of epidemiologic investigation. Clinically, the value of tuberculin test, though remarkable, is limited by the fact that its positivity is not necessarily a sign of active tuberculosis. The three control strategies of tuberculosis are: prompt identification and correct management of cases, vaccination, prophylaxis. The latter, that in most cases is performed with isoniazid (300 mg/daily for 12 months) is indicated in the following situations: subjects with > 5 mm tuberculin test; recent contacts with patients with infective tuberculosis; chest X-ray indicative for old fibrotic lesions, HIV infection; subjects with > 10 mm tuberculin test: HIV-negative drug-addicts; clinical conditions at high risk for tuberculosis (e.g. silicosis, hematologic malignancy, iatrogenic immunosuppression).     

Effect of physical activity as a caddie on ultrasound measurements of the Os calcis: a cross-sectional comparison

Human epidermal keratinocytes synthesize, secrete, and degrade acetylcholine and use their cell-surface nicotinic and muscarinic cholinergic receptors to mediate the autocrine and paracrine effects of acetyl-choline. Because acetylcholine modulates transmembrane Ca2+ transport and intracellular metabolism in several types of cells, we hypothesized that cholinergic agents might have similar effects on keratinocytes. Nicotine increased in a concentration-dependent manner the amount of 45Ca2+ taken up by keratinocytes isolated from human neonatal fore-skins. This effect was abolished in the presence of the specific nicotinic antagonist mecamylamine, indicating that it was mediated by keratinocyte nicotinic acetylcholine receptor(s). The sequences encoding the alpha 5 and alpha 7 nicotinic receptor subunits were amplified from cDNA isolated from cultured keratinocytes. These subunits, as well as the alpha 3, beta 2, and beta 4 subunits previously found in keratinocytes, can be components of Ca(2+)-permeable nicotinic receptor channels. To learn how activation of keratinocyte nicotinic receptors affected the rate of cell differentiation, we measured the nicotinic cholinergic effects on the expression of differentiation markers by cultured keratinocytes. Long-term incubations with micromolar concentrations of nicotine markedly increased the number of cells forming cornified envelopes and the number of cells staining with antibodies to suprabasal keratin 10, transglutaminase type I, involucrin, and filaggrin. The increased production of these differentiation-associated proteins was verified by Western blotting. Because nicotinic cholinergic stimulation causes transmembrane Ca2+ transport into keratinocytes, and because changes in concentrations of intracellular Ca2+ are known to alter various keratinocyte functions, including differentiation, the subcellular mechanisms mediating the autocrine and paracrine actions of epidermal acetylcholine on keratinocytes may involve Ca2+ as a second messenger.     

EGF-induced redistribution of erbB2 on breast tumor cells: flow and image cytometric energy transfer measurements

The effects of the CCK(B) antagonists, CAM1028 and CI988 and a CCK(A) antagonist, CAM1481, were studied on the anxiety-related behaviour produced by withdrawal from chronic ethanol treatment, using the elevated plus maze. Cessation of chronic ethanol administration produced a profile, in both mice and rats, consistent with increase in anxiety-related behaviour. In mice, SC administration of CAM1028 or CI988 reduced the decrease in the time spent on the open arms, the number of entries into these arms and the increases in the latencies to first open arm entry, after withdrawal from the ethanol treatment. The increases in stretched attend postures and head dips from the closed arms and the central square seen during the withdrawal phase, were also decreased by the CCK(B) antagonists, but the decreases in the number of rears and in general activity were unaffected. The doses of CAM1028 and CI988 tested were 0.1 and 1 mg/kg; for some of the withdrawal-induced changes in behaviour only the 1 mg/kg dose was effective. In contrast, the CCK(A) antagonist, CAM1481, at the same doses, had little effect on the anxiety-related behaviour produced by withdrawal from chronic ethanol treatment, although it did decrease the changes in the number of rears and the head dipping behaviour. In rats, the majority of the changes produced by withdrawal from chronic ethanol treatment were decreased by CAM1028 at 1 mg/kg, although the decreases in open arm entries, rearing behaviour and in overall activity were unaffected. CAM1028, CI988 and CAM1481 had no effects on the behaviour of control mice or rats in the plus-maze. The results show that CCK(B) antagonists were effective in decreasing the majority of the anxiogenic effects of withdrawal from chronic ethanol treatment.