Femtosecond infrared spectroscopy of channelrhodopsin-1 chromophore isomerization.

Vibrational dynamics of the retinal all-trans to 13-cis photoisomerization in channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) was investigated by femtosecond visible pump mid-IR probe spectroscopy. After photoexcitation, the transient infrared absorption of C-C stretching modes was detected. The formation of the 13-cis photoproduct marker band at 1193 cm(-1) was observed within the time resolution of 0.3 ps. We estimated the photoisomerization yield to (60 ± 6) %. We found additional time constants of (0.55 ± 0.05) ps and (6 ± 1) ps, assigned to cooling, and cooling processes with a back-reaction pathway. An additional bleaching band demonstrates the ground-state heterogeneity of retinal.

Ultrafast electronic and vibrational dynamics in brominated aluminum corroles: Energy relaxation and triplet formation.

We combined femtosecond (fs) VIS pump-IR probe spectroscopy with fs VIS pump-supercontinuum probe spectroscopy to characterize the photoreaction of the hexacoordinated Al(tpfc-Br8)(py)2 in a comprehensive way. Upon fs excitation at ∼400 nm in the Soret band, the excitation energy relaxes with a time constant of (250 ± 80) fs to the S2 and S1 electronic excited states. This is evident from the rise time of the stimulated emission signal in the visible spectral range. On the same time scale, narrowing of broad infrared signals in the C=C stretching region around 1500 cm(-1) is observed. Energy redistribution processes are visible in the vibrational and electronic dynamics with time constants between ∼2 ps and ∼20 ps. Triplet formation is detected with a time constant of (95 ± 3) ps. This is tracked by the complete loss of stimulated emission. Electronic transition of the emerging triplet absorption band overlaps considerably with the singlet excited state absorption. In contrast, two well separated vibrational marker bands for triplet formation were identified at 1477 cm(-1) and at 1508 cm(-1). These marker bands allow a precise identification of triplet dynamics in corrole systems.

[Computer aided dosage management of phenprocoumon anticoagulation therapy. Clinical validation]. / Rechnergestützte Dosierung in der Antikoagulationstherapie mit Phenprocoumon. Validierung für den klinischen Einsatz.

UNLABELLED: A recently developed multiparameter computer-aided expert system (TheMa) for guiding anticoagulation with phenprocoumon (PPC) was validated by a prospective investigation in 22 patients. The PPC-INR-response curve resulting from physician guided dosage was compared to INR values calculated by "twin calculation" from TheMa recommended dosage. Additionally, TheMa was used to predict the optimal time to perform surgery or invasive procedures after interruption of anticogulation therapy. RESULTS: Comparison of physician and TheMa guided anticoagulation showed almost identical accuracy by three quantitative measures: Polygon integration method (area around INR target) 616.17 vs. 607.86, INR hits in the target range 166 vs. 161, and TTR (time in therapeutic range) 63.91 vs. 62.40 %. After discontinuation of anticoagulation therapy, calculating the INR phase-out curve with TheMa INR prognosis of 1.8 was possible with a standard deviation of 0.50 ± 0.59 days. CONCLUSION: Guiding anticoagulation with TheMa was as accurate as Physician guided therapy. After interruption of anticoagulant therapy, TheMa may be used for calculating the optimal time performing operations or initiating bridging therapy.

Quantitative determination of metabolization dynamics by a real-time 13CO2 breath test.

We have developed a flow-through fast liver investigation packet (FLIP) to quantitatively measure the amount of exhaled (13)CO(2), as a result of liver metabolization processes. The FLIP system allows investigation of every single breath in real time. Line width variations due to interactions with other gas components disturb traditional measurements and limit their sensitivity to the ppm range. Detection of the complete breath volume and the spectrally fully resolved line shape allows sensitivity in the ppb range with a standard deviation of approximately 80 ppb, a prerequisite to quantitatively analyze liver metabolization processes.

Revealing anharmonic couplings and energy relaxation in DNA oligomers by ultrafast infrared spectroscopy.

The identification and characterization of NH 2 hydrogen-bonded stretching vibrations [nu(NH 2)] in DNA oligomers is usually hampered by the all-dominating absorption of the water stretching band in the spectral range of 3050-3600 cm(-1). Here, we use the two-color IR pump-probe technique to overcome the limitations of linear absorption spectroscopy by exciting adenine-thymine (A-T) oligomer vibrations in the fingerprint region and analyzing induced transient spectral changes in the nu(NH2) spectral region. These transient changes are related to anharmonic couplings to the modes excited in the fingerprint region and to modes populated by intra- and intermolecular energy redistribution and relaxation. The combination of calculated anharmonic coupling parameters and experimental transient IR data allows the assignment of a transition at 3215 cm(-1) to the nu(NH2) vibration of adenine in dA(20)-dT(20) DNA oligomers.

Identification of a putative p53 binding sequence within the human mitochondrial genome.

A small fraction of the total cellular amount of nuclear transcription factor p53 seems to be located at and within mitochondria. Transcription factors of the steroid receptor superfamily that, like p53, lack a classical mitochondrial leader sequence are nonetheless imported into mitochondria where they regulate mtDNA transcription through binding to specific recognition sequences. Here, we examined seven candidate sequences from the human mitochondrial genome with similarity to the consensus p53 binding motif. Two imperfect half-sites at coordinate 1553 with homology to the nuclear IGF-BP3 box A binding sequence are demonstrated to confer responsivity to p53 and the p53 relatives p73alpha and beta in the context of the cell nucleus. Mitochondrial p53 may thus bind directly to mtDNA and, perhaps, be involved in the regulation of mitochondrial transcription/replication.

Increased susceptibility of a carrier of X-linked chronic granulomatous disease (CGD) to Aspergillus fumigatus infection associated with age-related skewing of lyonization.

Chronic granulomatous disease (CGD) is an inherited disorder characterized by the inability of phagocytes to generate normal amounts of superoxide (O2-), leaving patients susceptible to life-threatening infections. It was previously assumed that once carriers of the X-linked form of CGD were found to have 30% or more of functionally normal neutrophils, they would be free of risk for infection because the lyonization ratio was believed to be constant. Our report strongly contradicts this assumption. A 45-year-old X-CGD carrier had approximately 40% of normal neutrophils in her peripheral blood at age 21 years. Recently, she contracted a life-threatening pulmonary infection with Aspergillus fumigatus. After recovery, the ratio of normal-to-nonfunctional neutrophils was re-evaluated. She was found to have only 6-8% of normal neutrophils, suggesting that a striking decrease in the number of normal cells over the past 25 years was the reason for an increased susceptibility to Aspergillus infection. We conclude that age-related acquired skewing of the lyonization ratio can result in an increased susceptibility to life-threatening infections in X-CGD carriers.

A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection.

The immunogenetic basis of severe infections caused by bacille Calmette-Guérin vaccine and environmental mycobacteria in humans remains largely unknown. We describe 18 patients from several generations of 12 unrelated families who were heterozygous for 1 to 5 overlapping IFNGR1 frameshift small deletions and a wild-type IFNGR1 allele. There were 12 independent mutation events at a single mutation site, defining a small deletion hotspot. Neighbouring sequence analysis favours a small deletion model of slipped mispairing events during replication. The mutant alleles encode cell-surface IFNgamma receptors that lack the intra-cytoplasmic domain, which, through a combination of impaired recycling, abrogated signalling and normal binding to IFNgamma exert a dominant-negative effect. We thus report a hotspot for human IFNGR1 small deletions that confer dominant susceptibility to infections caused by poorly virulent mycobacteria.

Microheterogeneity of serum glycoproteins and their liver precursors in patients with carbohydrate-deficient glycoprotein syndrome type I: apparent deficiencies in clusterin and serum amyloid P.

Serum and liver protein patterns were studied, respectively, in 5 patients (serum) and 1 patient (liver) with carbohydrate-deficient glycoprotein syndrome (CDGS) type I by high-resolution two-dimensional electrophoresis (2-DE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pattern of serum glycoproteins in all 5 patients presented abnormal trains of isoforms with decreased mass (delta molecular weight 3000) and all showed a cathodal shift. Two-dimensional electrophoresis and SDS-PAGE mass analysis of transferrin, alpha1 -antitrypsin, haptoglobin beta-chain, and alpha1-acid glycoprotein after neuraminidase and N-glycosidase F treatments demonstrated that the additional trains of the isoforms found in CDGS type I contain homologous species of isoforms. Some of them still showed charge differences, and all still contained glycans except for transferrin, with some unusual nonglycosylated isoforms. In addition, deficiencies in clusterin and serum amyloid P, not described so far, have been found in all 5 patients. The two-dimensional pattern of immunodetected precursors of serum proteins in liver cells from 1 patient with CDGS showed abnormal low-mass precursors and the absence of the precursors normally found in controls. These results suggest that these abnormal precursors accumulate during the early oligosaccharide processing of the nascent protein-bound oligosaccharides and that glycoprotein precursors undergo an altered intracellular transport while the post-translational processing along the normal pathway is still apparently functioning in patients with CDGS.

p53 expressions: predicting recurrence and second primary tumors in head and neck squamous cell carcinoma.

BACKGROUND: The survival rate for head and neck squamous cell carcinoma remains poor despite therapeutic advances over the last two decades. For patients with disease confined to the head and neck, there are two major and biologically distinct patterns of treatment failures after definitive therapy: recurrence of primary disease and development of second primary tumors. Understanding the biological basis of patterns of treatment failure after definitive therapy is needed to guide the development of adjuvant treatment and strategies to prevent second primary tumors. PURPOSE: To determine whether expression of the p53 protein has prognostic significance and/or is associated with patterns of treatment failure, we examined protein expression in primary tumor specimens of patients with head and neck squamous cell carcinoma. METHODS: Immunohistochemical analysis with a monoclonal antibody (DO7) specific for p53 protein was used to detect expression of the protein in formalin-fixed, paraffin-embedded tumor samples from 69 head and neck cancer patients treated with definitive local therapy (surgery and/or radiotherapy) between January 1980 and October 1983 at The University of Texas M. D. Anderson Cancer Center. We quantitated p53 protein expression and assessed its association with duration of patient survival, patterns of treatment failure (recurrence of primary tumor and development of second primary tumor), and other clinical parameters. All reported P values resulted from two-sided statistical tests. RESULTS: We found detectable levels of p53 protein expression in the tumor cell nuclei of 41 of 69 patients. Thirty-six (52%) of 69 patients whose tumors exhibited p53 protein expression in greater than or equal to 10% of the cell nuclei were grouped as p53 positive, and 33 (48%) of 69 patients whose tumors exhibited less than 10% nuclear expression were groups as p53 negative. The clinical characteristics of the patients in the p53-positive, and p53-negative groups were well balanced. Overall survival was significantly lower, and the times to tumor recurrence, to second primary tumors, and to any treatment failure were significantly shorter in the p53-positive group that in the p53-negative group (P=.0002, P=.047, P=.003, and P=.0009, respectively), mainly because the p53 positivity was associated with earlier development of tumor recurrence and second primary tumors. The rate of second primary tumor development per person per year was also significantly higher in the p53-positive group that in the p53-negative group. By use of multivariate analysis according to the Cox regression model, p53 expression status was identified as the most significant predictor of overall survival duration (P=.007), time to tumor recurrence (P=.053), time to second primary tumors (P=.035), and time to any treatment failure (P=.004). CONCLUSIONS: The expression of p53 protein in primary head and neck squamous cell carcinoma was significantly predictive of shorter survival because of its association with earlier development of both tumor recurrence and second primary tumors. Thus, p53 expression may be a valuable marker for identifying individuals at high risk of developing a recurrence of primary disease and second primary tumors who may benefit from adjuvant therapy and chemoprevention after definitive local therapy.

[Diagnosis and nosology of glycanosis CDG ("carbohydrate deficient glycoprotein syndrome")]. / Diagnostik und Nosologie der Glykanose CDG ("Carbohydrate-deficient glycoproteinsyndrome").

BACKGROUND: The "Carbohydrate-deficient glycoprotein syndrome" is a recently discovered inborn error of complex carbohydrate metabolism. The disease involves a number of organ systems and various deficient glycoproteins. An abnormal isoform of serum transferrin is of diagnostic value. METHODS: We analysed the glycoprotein alpha-1-antitrypsin of two affected infants and their clinically healthy parents using high resolution isoelectric focusing technique. Besides normal isoforms of alpha-1-antitrypsin, we found an abnormal cathodic isoform ("CDG-alpha-1-antitrypsin") which represented almost half of the total amount of alpha-1-antitrypsin of the patients. RESULTS: This new marker-glycoprotein suggests a defect of the production of biantennary and of triantennary N-glycans during an early step of their synthesis, resulting in monoantennary N-glycans. Also this marker-glycoprotein seems to be a specific biochemical diagnostic tool for discovering glycanosis CDG (Carbohydrate-deficient glycoprotein syndrome). The mode of inheritance is probably incomplete autosomal dominant. The same genetic defect of N-glycan synthesis may be present in more than one type of the hybrid molecule glycoprotein, and was also found in transferrin, resembling a "genetic back-pack", that might explain the multitude of clinical symptoms. CONCLUSION: In view of these findings, we present novel systematics of those diseases that are due to inborn errors of N-glycan synthesis, and which we suggest to call "glycanoses".

The incidence of second primary tumors in long-term survivors of small-cell lung cancer.

PURPOSE AND METHODS: A review of 446 patients who were enrolled consecutively in small-cell lung cancer (SCLC) protocols was performed to identify in long-term survivors the frequency of new primary tumors and their clinical impact. RESULTS: Forty-seven patients (10.5%) were identified to be free of disease at 2 years. Second primary tumors (SPTs) were diagnosed in 14 patients. The overall risk for developing an SPT was 10.3% per person-year. Actuarial risk at 8 years was 50.3% for an SPT. CONCLUSIONS: In this review, SCLC showed one of the highest incidences of SPTs reported in aerodigestive tract malignancies. A long-term survivor was more likely to have an SPT than a relapse of SCLC. Consequently, the odds of death from an SPT compared with that from a relapse increased sharply from 1:13 within 4 years from diagnosis to 8:1 afterwards. Long-term survivors of SCLC would be excellent candidates for chemoprevention trials.

Chemoprevention in head and neck cancer.

It is not a coincidence that the aerodigestive tract has proven such a fertile ground for the evaluation of chemoprevention. The incidence of second primaries and the concept of field cancerization made the need for prevention acute and showed the limitations of even the most curative of treatments. New and presumably less toxic agents are being evaluated; chemoprevention is being applied to asymptomatic populations at higher risk for lung, colon, breast, and other neoplasias. Biomarkers may be instrumental in rapid development of these new clinical applications, but much is still to be done. Yet unanswered is whether suppression of premalignant lesions will ultimately decrease cancer incidence. Survival in the second primary prevention trial has not thus far shown a significant improvement and toxicities were significant. Many questions remain in the study of chemoprevention; head and neck cancer provides a conducive model in which these answers might be found.

Variation of alpha 1-antitrypsin glycoprotein microheterogeneity in hepatic postresuscitation disease.

Microheterogeneity of the glycoprotein alpha 1-antitrypsin has been investigated sequentially by high resolution isoelectric focusing in a child with the proteinase inhibitor MS phenotype after near-drowning. A band-splitting with additional cathodal fractions exhibited migration from the most cathodic to the anodic positions of the glycoprotein isoforms in the course of post-resuscitation disease. The pattern may reflect the time- and stage-dependent hypoxic and post-hypoxic effects on hepatocellular metabolic zonation.