Long lifetimes of quantum-dot intersublevel transitions in the terahertz range.

Carrier relaxation is a key issue in determining the efficiency of semiconductor optoelectronic device operation. Devices incorporating semiconductor quantum dots have the potential to overcome many of the limitations of quantum-well-based devices because of the predicted long quantum-dot excited-state lifetimes. For example, the population inversion required for terahertz laser operation in quantum-well-based devices (quantum-cascade lasers) is fundamentally limited by efficient scattering between the laser levels, which form a continuum in the plane of the quantum well. In this context, semiconductor quantum dots are a highly attractive alternative for terahertz devices, because of their intrinsic discrete energy levels. Here, we present the first measurements, and theoretical description, of the intersublevel carrier relaxation in quantum dots for transition energies in the few terahertz range. Long intradot relaxation times (1.5 ns) are found for level separations of 14 meV (3.4 THz), decreasing very strongly to approximately 2 ps at 30 meV (7 THz), in very good agreement with our microscopic theory of the carrier relaxation process. Our studies pave the way for quantum-dot terahertz device development, providing the fundamental knowledge of carrier relaxation times required for optimum device design.

Fungal endogenous rhythms expressed by spiral figures.

Culture zonations in two fungi, Nectria cinnabarina and Penicillium diversum, are expressions of endogenous rhythms. These culture zonations may take the form of either concentric rings or Archimedes' spirals. The rhythm in N. cinnabarina is noncircadian. The rhythm in P. diversum is relatively insensitive to temperature and has a period of approximately 24 hours. The lack of a demonstrable mechanism for phase shifting suggests that this rhythm may also be noncircadian.

Evolutionary conserved mechanism of transcriptional repression by even-skipped.

Even-skipped (Eve) is a transcriptional repressor involved in segment formation in Drosophila melano-gaster. In order to gain further insights into the mechanism of action of Eve we tested whether it would function as a transcriptional repressor in mammalian cells. We found that Eve was indeed a potent repressor in two different mammalian cell types and at several promoters. In vitro transcription assays confirmed that Eve directly represses transcription initiation when specifically targeted to a promoter. We also found that, unlike the case with transcriptional activators, Eve does not repress transcription synergistically. Analysis of the effect of Eve on preinitiation complex assembly in a crude HeLa cell nuclear extract demonstrated that the Eve repression domain functions by preventing the assembly of TFIID with the promoter. Our data support the hypothesis that Eve contains an active repression domain that functions specifically to prevent preinitiation complex formation.

Regulation of the Wilms' tumour suppressor protein transcriptional activation domain.

The Wilms' tumour suppressor protein WT1 contains a transcriptional regulatory domain that can either activate or repress transcription depending upon its cellular environment. The mechanistic basis for this dichotomy is unclear however. Here, we dissect the transcriptional regulatory domains of WT1. We find that a region within the domain of WT1 attributed to transcriptional repression is a potent suppressor of the activation domain at several promoters and in different cell types. In vitro transcription analysis suggests that the mechanism of suppression of the activation domain occurs at the level of transcription initiation. Furthermore we find that the WT1 suppression domain is able to inhibit a heterologous activation domain when fused in cis. Dissection of this domain resulted in the delineation of a 30 amino acid region that was sufficient to confer suppression of a transcriptional activation domain both in vivo and in vitro. Additionally, we find that the WT1 transcriptional activation domain interacts with the general transcription factor TFIIB and that this interaction is not affected by the suppression domain. Taken together, these studies suggest that the suppression domain of WT1 interacts with a cosuppressor protein to mediate inhibition of the WT1 transcriptional activation domain.

Isolation, characterisation and growth-related changes of an HMG-like protein from microplasmodia of Physarum polycephalum.

An alanine, lysine and glutamic acid-rich nuclear protein (P2) of Mr approximately 19,500 co-extracts with the histones from nuclei of Physarum polycephalum when using the CaCl2 method for histone extraction [1] and was found to have the composition previously ascribed to a putative histone H1(0) isolated from microplasmodia using 5% PCA (Yasuda, H., Mueller, R.D., Logan, K.A. and Bradbury, E.M. (1986) J. Biol. Chem. 261, 2349-2354). P2 has very similar electrophoretic properties to chicken erythrocyte histone H5, calf thymus histone H1(0) and the Physarum HMG-like protein AS-2, but does not appear to be immunologically or structurally similar to H5 or H1(0). An increase in the abundance of P2 was observed during exponential growth in microplasmodia, reaching an approximately 1:1 ratio with histone H1 by 48 h of culture. Standard amino acid analysis and NMR show that P2 is more HMG-like than H1-like and CD measurements demonstrated that P2 contains only 5% secondary structure in its maximally structured state and is, therefore, essentially unstructured under in vivo conditions. Also possible clustering of acidic residues is detected using CD and may be of functional significance. Analysis of post-translational modification of P2 shows that it is phosphorylated at up to three sites as isolated from immature spherules. The relationship of P2 to the HMG family of proteins and AS-2 is discussed.

Differential synthesis of histone H1 during early spherulation in Physarum polycephalum.

H1 and P2 (an H1 degree/HMG-like protein) accumulate during exponential growth of Physarum microplasmodia (unpublished results), indicating that these proteins may play a role in differentiation (spherulation). To test this hypothesis, pulse labelling using [14C]lysine was used to determine whether any differential histone synthesis occurs during salts-induced spherulation. A peak in the uptake of [14C]lysine into microplasmodia was detected between 12 and 24 h following salts-induction. During the same interval, incorporation of label into the CaCl2-extracted histones occurred, with H1 being synthesised at approx. 3 times the level of the core histones and P2. Densitometry of SDS-PAGE gels showed that high levels of H1 were maintained up to 40 h in salts medium, beyond the observed peak in synthesis. The synthesis and accumulation of high levels of H1 during early spherulation indicates a role for this histone in the initiation and maintenance of a transcriptionally inactive differentiated state.

Selective depletion and reconstitution of nucleosome core particles.

A method for the complete and specific removal of histones H2A and H2B from nucleosome core particles is presented. Reconstitution of the separated products of depletion form a particle which has the same structure as native core particles as judged by a number of physical and biochemical criteria. The technique described also minimises the possibility of the formation of reconstituted core particles with different histone stoichiometries. These experiments are important as they demonstrate a procedure which can be extended to prepare core particles with selectively deuterated components while maintaining complete integrity of structure. When prepared, and studied by neutron scattering, selectively-deuterated core particles can give detailed information with respect to the relative positions and structure of the histone fractions within the core particle.

Polyamines permit the preparation of stable Physarum core particles which have a structure similar to those from higher eukaryotes.

The inherent instability of Physarum nucleosome core particles prepared by micrococcal nuclease digestion in Na+/Ca2+ buffers can be overcome by the addition of 0.15 mM spermine and 0.5 mM spermidine. Neutron scattering, circular dichroism, nuclease digestion and thermal denaturation studies carried out on these stable monosomes show them to be very similar to those obtained from higher eukaryotes.

Long-term prostaglandin E1 therapy in congenital heart defects.

Seventeen neonates received an intravenous infusion of prostaglandin E1 for an average of 39 days (range 8 to 104). Seven (group 1) had transposition of the great arteries with no ventricular septal defect or a small one; eight (group 2) had ductus-dependent pulmonary flow (pulmonary atresia or stenosis in six and tricuspid atresia in two); and two (group 3) had aortic coarctation, one with no ventricular septal defect, the other with ventricular septal defect, isthmus hypoplasia and descending aortic flow supplied mainly by the ductus. An increase in the arterial partial pressure of oxygen (PO2) was seen in groups 1 and 2. Six patients from group 1 and two from group 2 developed heart failure; cortical hyperostosis of long bones was seen in three patients from group 1 and three from group 2; one from group 1 had refractory diarrhea. Other side effects seen at the beginning improved as the rate of infusion diminished. In group 3, the patient with complex coarctation had a decrease in blood pressure in the arms, an increase in pressure in the legs and restoration of renal function; in the patient with no ventricular septal defect, heart failure worsened during therapy. Histologic changes seen in three ductus were attributed to the closing process. When delaying surgery in selected ill infants with heart defects is deemed advantageous, long-term infusions of prostaglandin E1 are feasible.

The cost of steroid-free protocols in primary cadaver kidney transplantation.

Steroid-free immunosuppression regimens have been enjoying recent success in clinical transplantation. The use of antibodies required for such protocols can be an economic burden. We proposed to study their cost in our center. This retrospective study involved 147 consecutive patients subjected to 4 protocols of immunosuppression. The first received triple therapy. The second group received induction with basiliximab, whereas the third received Basiliximab plus cyclosporine (CSA) plus mycophenolate mofetil (MMF), and the fourth received Thymoglobulin plus CSA plus MMF in conjuction with only 4 days of steroid. Rejection episodes were treated with Solumedrol. Six-month charges were obtained from computerized records of the finance department, the in-house laboratories, and the transplantation service registry. All charges were expressed in 2004 dollars. Statistical analyses were obtained using chi-square, analysis of variance (ANOVA) and Kaplan-Meier tests. The 4 groups were similar with regard to donor and/or recipient gender, race, panel reactive antibodies, cold ischemia, dialysis requirements length of stay and readmission, graft survival, and function. Charges were significantly higher in the last 2 groups as compared with triple therapy.

Hemispheric asymmetries in schizophrenia.

The growing literature on hemispheric asymmetries in schizophrenic populations is critically reviewed. Studies of lateral asymmetries in schizophrenics are discussed which have employed a wide range of methodologies, including assessment of motor, sensory, electrophysiological, neuropsychological, and structural abnormalities. This literature is discussed in relation to two theoretical viewpoints, one emphasizing impaired functioning of the corpus callosum, and the other positing left hemisphere overactivation and dysfunction in schizophrenic populations. It is concluded that the hypothesis of impaired callosal function has not been adequately tested because of methodological problems, the most serious of which is the failure to show differential deficit. The hypothesis of left hemisphere abnormality has gained consistent support, although methodological problems were noted. This research suggests a structural locus for schizophrenic pathology which is consistent with the symptomatology of the disorder, and provides avenues for further research.

The lysine-rich H1 histones from the slime moulds, Physarum polycephalum and Dictyostelium discoideum lack phosphorylation sites recognised by cyclic AMP-dependent protein kinase in vitro.

Calcium chloride-extracted histones were prepared from nuclei of the slime moulds, Physarum polycephalum and Dictyostelium discoideum, and phosphorylation by purified preparations of cyclic AMP-dependent protein kinase (cAMP-d PK) and growth-associated H1 histone kinase (HKG) examined and compared. Among the major histone fractions and other proteins in the two preparations, the H1 histones from both organisms were found to be effective and exclusive substrates for HKG. cAMP-d PK, which phosphorylates mammalian H1 histone and certain, in particular H2B, of the mammalian core histones, phosphorylated several of the core histones from both slime moulds but did not phosphorylate H1 histone from either. The slime mould H1s remained ineffective substrates for cAMP-d PK even after extensive alkaline phosphatase treatment of the histone preparations. Additional studies demonstrated that the lack of slime mould H1 phosphorylation by cAMP-d PK was not due to competition of the H1 molecules with the core histones for the kinase. Our studies suggest that H1 histones from these organisms, whilst clearly containing sites for phosphorylation by HKG, apparently lack phosphorylation sites recognised by cAMP-d PK. Thus, the mediation of specific nuclear functions by cAMP-dependent phosphorylation of H1 in higher organisms may not occur or be required in these lower eukaryotes.

Comparison of African-American and white persons with Alzheimer's disease on language measures.

We assessed 11 African-American and 32 white subjects with early to midstage AD using seven measures (the Boston Naming Test, Peabody Picture Vocabulary Test-Revised, Shortened Token Test, a modified Reporter's Test, two subtests of a shortened Rey Auditory Verbal Learning Test, and selected stimuli from the Test of Problem Solving). There were no ethnic differences, with Mini-Mental State Examination score and education accounting for most of the variance between ethnic groups. However, white subjects tended to score higher than African-Americans on five of the seven measures. African-Americans tended to perform better on the Test of Problem Solving, a measure of the pragmatic use of language. Although these preliminary findings suggest no test bias for ethnicity, the trends indicate that language measures should continue to be examined for ethnic differences in larger samples.

Evaluation of cardiovascular risk for renal transplantation in diabetic patients.

Cardiovascular disease contributes in a major way to morbidity and mortality in diabetic patients with end-stage renal disease. Sixty patients with type I diabetes were evaluated prior to renal transplantation to determine the risk of cardiovascular complications. On the basis of results of thallium stress testing and/or cardiac catheterization, each patient was assigned to one of five categories. There were no cardiovascular events in the seven patients who had negative results on stress testing. Of the remaining 53 patients, all of whom underwent cardiac catheterization, 30 had normal coronary arteries. None of these 30 patients had any cardiac morbidity, and the two deaths that occurred in this group were not attributable to cardiac causes. Significant coronary artery disease was present in 38 percent of the patients. The overall mortality rate was 5.4 percent in those patients without coronary artery disease and 43.5 percent in those with the disease. In addition, the mortality rate in patients with coronary disease classified as severe was 62 percent, whereas it was 20 percent in those categorized as having moderate disease. The data indicate that patients with diabetes and end-stage renal disease who are at highest risk for cardiovascular events can be identified, and these patients probably should not undergo renal transplantation.

Influence of DR matching in cadaveric renal transplants performed with cyclosporine. A retrospective analysis of one center.

A retrospective analysis of 300 consecutive cadaveric renal allografts performed at our institution between August 1, 1981, and December 1, 1983, was performed to evaluate the influence of DR typing on graft and patient survival. All patients were treated with low-dose steroids and cyclosporine as the only means of immunosuppression. The group included 246 primary graft recipients and 54 retransplants. DR information was available on the donor and the recipient in 225 of these patients, and it was unavailable on the donor and/or recipient in 75 patients. In 49% of the cases in which information was available, 2 alleles were identified in the donor and in the recipient; in the remainder only 1 allele was identified in either the donor or recipient. The results were analyzed according to HLA/DR match and mismatch. Twelve-month graft survival for the 2-DR-match recipients was 67%, versus 78% for the 1-DR match and 76% for the O-DR match. These differences were not significant. For the O-DR mismatch, the one-year actuarial graft survival was 74%, for the 1-DR mismatch 78%, and for the 2-DR mismatch 79%. Again, there was no significant difference. There was no impact of DR matching on patient or graft survival up to 18 months. Additionally, no difference was found in any of the groups regarding the number of treated rejection episodes per patient or the amount of steroid received per patient at the end of a year. These results suggest that cyclosporine negates the effect of DR matching in cadaveric renal transplantation.

Increased frequency of alloantigen-reactive helper T lymphocytes is associated with human cardiac allograft rejection.

Endomyocardial biopsy (EMB) is the standard method of monitoring heart transplant recipients for the development of allograft rejection. To date, noninvasive methods to detect cardiac allograft rejection have lacked adequate sensitivity and specificity for wide clinical application. In this study, limiting dilution analysis (LDA) was used to quantitate the number of donor alloantigen-reactive helper T lymphocytes (HTLs) in the peripheral blood of cardiac transplant recipients. Cadaveric donor splenocytes were cryopreserved, providing a source of donor alloantigenic stimulation for these assays. Peripheral blood mononuclear cells were harvested from cardiac transplant recipients before transplantation and at the time of EMB. LDA of donor-reactive HTLs was conducted simultaneously on all time points to minimize experimental variation, and these data were related to EMB scores. Frequencies of donor-reactive HTLs in pretransplant samples were highly variable, ranging from 1/1381 to < 1/200,000, and correlated poorly with the degree of HLA disparity. During episodes of moderate rejection, donor-specific HTL frequencies increased an average of 6 times their post-transplant baseline frequency. Additionally, 10-fold increases in HTL frequencies were seen preceding EMB-diagnosed rejection in several individuals. These data indicate that episodes of allograft rejection are associated with increases in the number of circulating donor-reactive HTL which are frequently detected before the development of histologically defined rejection. Thus, monitoring HTL frequencies may serve as a non-invasive method for detecting and predicting cardiac allograft rejection. Furthermore, this assay may provide a valuable means of assessing the in vivo efficacy of various immunosuppressive therapies.

4-halo-3-hydroxyanthranilic acids: potent competitive inhibitors of 3-hydroxy-anthranilic acid oxygenase in vitro.

The mechanism of action of three potent inhibitors of 3-hydroxyanthranilic acid oxygenase (3HAO), the enzyme responsible for the production of the endogenous excitotoxin quinolinic acid, was examined in vitro. Using either liver homogenate or purified 3HAO, and following the rapid synthesis of the immediate enzymatic product alpha-amino-beta-carboxymuconic acid omega-semialdehyde spectrophotometrically, 4-halogenated (F, Cl, Br) 3-hydroxyanthranilic acids were found to inhibit enzymatic activity in a reversible fashion. Because of the very tight binding of the drugs to 3HAO, reversibility was detected only after warming the protein-inhibitor complexes at 37 degrees. Further studies showed that enzyme inhibition was competitive in nature (apparent Ki values: 190, 6 and 4 nM for the F-, Cl- and Br-compounds, respectively), and suggested that the drugs are metabolized by the enzyme. Specific, reversible, and tightly binding 3HAO inhibitors can be expected to become valuable tools for the study of quinolinate neurobiology. The drugs could also be of interest for the diagnostics and therapeutics of brain diseases which have been speculatively linked to a pathological overabundance of quinolinic acid.

Studies of structure and mechanism in acetonitrile chemical ionization tandem mass spectrometry of polyunsaturated fatty acid methyl esters.

Recently it has been shown that acetonitrile chemical ionization tandem mass spectrometry (CI-MS/MS) is a rapid, on-line means to determine double bond position in fatty acid methyl esters (FAME). The mechanism of this gas phase condensation reaction has been studied. Evidence of the (1-methyleneimino)-1-ethenylium ion (m/z 54), formed upon the reaction of acetonitrile with itself, adding across the double bond in a [2 + 2] cycloaddition reaction is observed. When this nascent complex undergoes collision-induced dissociation, two diagnostic ions emerge. One of these ions results from loss of the hydrocarbon end of the FAME, whereas the other ion results from loss of the methyl ester end, and when considered together, the diagnostic ions localize the positions of the double bonds in the FAME. Several labeling and MS/MS/MS experiments on the two diagnostic ions were performed to determine a plausible fragmentation mechanism of the stable (1-methyleneimino)-1-ethenylium-FAME complex. The first generation product ions, or diagnostic ions, appear to be formed though a charge-driven mechanism, whereas the second generation product ions are formed via charge-remote fragmentations. Plausible mechanisms for the formation and subsequent dissociation of the diagnostic ions are presented for the monounsaturated, diunsaturated, and polyunsaturated (3 or more double bonds) FAME.

Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance.

Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding.