Light-Switchable Peptides with a Hemithioindigo Unit: Peptide Design, Photochromism, and Optical Spectroscopy.

This Minireview focuses on the hemithioindigo photoswitch and its use for the reversible control of three-dimensional peptide structure and related biological functions. Both the general design aspects and biophysical properties of various hemithioindigo-based chromopeptides are summarized. Hemithioindigo undergoes reversible Z→E photoisomerization after absorption of visible light. The unique ultrafast switching mechanism of hemithioindigo combines picosecond isomerization kinetics with strong double-bond torsion after light absorption, making it the ideal tool for instantaneous modulation of biological structure. Various inhibitors and model peptides based on hemithioindigo are described that can directly regulate biological signaling or allow the fastest events in peptide folding to be studied. Finally, a diverse range of chromopeptides with photoswitchable ß-hairpin structures based on azobenzenes, stilbenes, and hemithioindigo are compared to emphasize the unique properties of hemithioindigo.

Selective functionalization of patterned glass surfaces.

Tailored writing and specific positioning of molecules on nanostructures is a key step for creating functional materials and nano-optical devices, or interfaces for synthetic machines in various applications. We present a novel approach for the selective functionalization of patterned glass surfaces with functional probes of any nature. The presented strategy is optimized for imaging fluorophore labeled nanostructures for (single-molecule) fluorescence microscopy. The first step in the protocol is coating a glass surface, here a microscope cover slide, with a 60 nm thick diamond-like carbon film. Subsequently, the pattern is defined by either writing silicon oxide on the coating with a focused electron beam, or by etching the coating with a focused ion beam to expose the glass surface. Finally, the pattern is silanized and functionalized. We demonstrate the selective binding of organic fluorophores and imaging with high contrast, especially in total-internal-reflection mode. The presented approach is flexible and combines bottom-up assembly with high-resolution lithography on glass cover slides to precisely position and image functional molecules of any type.

Light-switchable hemithioindigo-hemistilbene-containing peptides: ultrafast spectroscopy of the Z → E isomerization of the chromophore and the structural dynamics of the peptide moiety.

Two hemithioindigo-hemistilbene (HTI) derivatives, designed to operate as structural switches in peptides, as well as two HTI peptides are characterized by ultrafast spectroscopy in the visible and the infrared. The two HTI switches follow the reaction scheme published for other HTI compounds with a picosecond excited state reaction (τ(1) ≈ 6 ps) and isomerization from Z to E with τ(2) = 13 and 51 ps. As compared to the isolated chromophores, the isomerization reaction is slowed down in the chromopeptides to τ(2) = 24 and 69 ps. For the smaller peptide containing 6 amino acids, the structural changes of the peptide moiety observed via the IR spectrum in the amide I band follow the isomerization of the molecular switch closely. In the larger cyclic chromopeptide, containing 20 amino acids and mimicking a ß-hairpin structure in the Z-form of the chromophore, the peptide moiety also changes its structure during isomerization of the chromophore. However, the IR spectrum at the end of the observation period of 3 ns deviates significantly from the stationary difference spectrum. These signatures indicate that strong additional structural changes, e.g., breaking of interchain hydrogen bonds, also occur on longer time scales.

Correlation of prostaglandin metabolizing enzymes and serum PGE2 levels with vitamin D receptor and serum 25(OH)2D3 levels in breast and ovarian cancer.

BACKGROUND: Vitamin D and its active form calcitriol have multiple effects in cancer cells, such as anti-proliferative effects, induction of apoptosis and cell cycle arrest. There is a link between vitamin D metabolism and inflammatory processes, which should be considered in cancer therapy. An association between these two types of metabolism is also observed in breast and ovarian cancer. These inflammatory processes are based on an increase of cyclooxygenase-2 (COX-2) activity. The current study aimed to evaluate the expression of prostaglandin-metabolising enzymes COX-2 and 15-hydroxyprostaglandin-dehydrogenase (15-PGDH) along with the vitamin D receptor (VDR) in benign and malignant breast and ovarian tissues. PATIENTS AND METHODS: VDR, COX-2, 15-PGDH and prostanoid receptor E2/E4 expression were measured in tissues by western blot analysis. Additionally, plasma 25(OH)(2)D(3) and PGE(2) levels were measured in healthy patients and cancer patients. RESULTS: We detected an elevated COX-2 and inversely a lowered VDR expression in cancer patients compared to healthy women. Breast cancer patients diagnosed during wintertime had a significantly lower serum level of 25(OH)(2)D(3); PGE(2) serum levels were higher in both types of cancer. CONCLUSION: These results support the idea of a link between prostaglandin and vitamin D metabolism in regards to their influences on breast and ovarian cancer.

Vitamin D-1alpha-hydroxylase and vitamin D-24-hydroxylase in benign and malign breast tissue.

BACKGROUND: It is known that 1,25(OH)2D3 can be metabolized to 1,24(OH)2D3 in breast tissue. This tissue-specific expression of 24-OHase may act as a pivotal link between vitamin D status (25(OH)D3 level) and the anticancer effects of 1,25(OH)2D3. Different expressions of the enzymes of vitamin D metabolism are found in breast cancer cells and tissues, and alternative splicing may play a role in biological functions and may cause tissue-specific variations. We describe the expression of vitamin D-1alpha-hydroxylase and vitamin D-24-hydroxylase in benign and malign breast tissues. We estimated that alternative splicing of the enzymes would lead to a catalytically dysfunctional product and may lead to a lower reduction of the target protein. MATERIAL AND METHODS: Expression of 1alpha-OHase and 24-OHase RNA and protein was assessed using a real-time polymerase chain reaction (RT-PCR) and on protein level by Western blot in benign and malign breast tissue samples. RESULTS: In breast cancer tissue the expression of 1alpha-OHase and 24-OHase were reduced significantly compared to benign breast tissue. CONCLUSION: The results described above do not support results of previous studies. Alternative splicing of 1alpha-OHase and 24-OHase may regulate the levels of active enzyme but is more likely due to different cell types in samples with the result of testing a variety of tissue samples not purified benign and malign breast cancer cells. The significance of smaller variants in cells has not been clarified either, but it is known that they are not able to use 25(OH)D3 as a substrate to generate 1,25(OH),D3.

Vitamin D-24-hydroxylase in benign and malignant breast tissue and cell lines.

Tissue-specific expression of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) and vitamin D-hydroxylase (24-OHase) may act as the pivotal link between 25-hydroxyvitamin D3 (25(OH)D3) serum levels and the anticancer effects of 1,25-dihydoxyvitamin D3 (1,25(OH)2D3) and alternative splicing of the enzymes may regulate their biological function. The expression of 24-OHase in cells and breast tissue was investigated and its splice variants were detected. The expression of 24-OHase RNA and protein was assessed by RT-PCR followed by Western blot. The expression of 24-OHase was reduced by about 57% in MCF-7 breast cancer cells, compared to MCF-10F benign breast cells. In the Western blot, a signal at 56 kDa was found and further bands were detected at 42 and 44 kDa. In the breast cancer tissue, the expression of 24-OHase was reduced by about 58% compared to benign tissue. However, in the Western blot, only one signal was found in the benign tissue at 56 kDa, while in malignant tissue, a further band was detected at 40 kDa. Alternative splicing of 24-OHase may lead to a catalytically dysfunctional enzyme and may lead to less reduction of the target protein.

Photochromic bis(thiophen-3-yl)maleimides studied with time-resolved spectroscopy.

The dynamics of the ring-closure reaction of three different bis(thiophen-3-yl)maleimides are investigated using ultrafast spectroscopy in the visible range. The structures of the molecules differ with respect to substitution of the thiophene ring and the maleimide. The experiments reveal reaction kinetics which point to the population of an excited electronic state for several nanoseconds. In the case of completely unsubstituted thiophene rings, a long excited-state lifetime (biexponential decay with 3 and 15 ns) can be observed. The remaining ultrafast absorption transients of this molecule are due to relaxational processes on the excited electronic potential energy surface. The ring-closure reaction has a small yield (<1%) and does not show up in the ultrafast absorption experiments. A dimethyl substitution of the thiophene ring results in completely different behavior: after transients related to relaxation in the excited electronic state, one finds pronounced absorption transients with tau = 16 ps which represent the partial decay of the excited electronic state and the formation of the ring-closed isomer. Another fraction of the emitting excited electronic state decays again on the few nanosecond time scale. The experiments suggest that the open isomer of the dimethyl-substituted imides exists in two conformations.

Tartrate-resistant acid phosphatase (TRAP) as a serum marker for bone resorption in breast cancer patients with bone metastases.

BACKGROUND: A novel immunoassay specific for the osteoclast-produced tartrate-resistant acid phosphatase TRAP isoform 5b was developed some years ago. By means of this assay, the usefulness of serum TRAP in monitoring the response to palliative treatment with clodronate in breast cancer patients with bone metastases was studied. Serum TRAP was examined for correlation with the activity of bone osteoclasts in these patients. MATERIALS AND METHODS: Seventeen patients took part in this study taking 1600 mg clodronate daily as a tablet for five months. Eleven of these patients were evaluated. RESULTS: TRAP activity correlated well with the grade of bone metastases and with the number of locations in the body. During the therapy with clodronate, TRAP activity in serum decreased. CONCLUSIONS: We conclude that the measurement of TRAP is useful in monitoring treatment with bisphosphonate clodronate in patients with bone metastatic breast cancer.

Feasibility and efficacy of chemotherapy with gemcitabine mono and with paclitaxel/mitoxantron in gynaecological cancers.

In this prospective study 32 patients with advanced gynaecologic tumours were treated with different schemes of chemotherapy: 15 received a combination of paclitaxel (100 mg/m2/week)/mitoxantron (6 mg/m2/every second week). Seventeen patients were treated with gemcitabine (100 mg/m2) in two different schedules, and the time of infusion was 2,2-3,3 hours or 30 minutes, respectively. Tolerability and efficacy were observed. The most common reason for reduction of the dosage or for cycle delay in the combined scheme was neutropenia. The response rate was 82%. The median overall survival was 30 weeks since beginning of the chemotherapy and 15 weeks after the last infusion. Gemcitabine in the shorter scheme led to a higher median dose rate. Toxic skin effects and hematological adverse events led to dose reduction and cycle delay in 90% of the infusions in the longer scheme. The response rate was 76%. The overall survival was one to 69 weeks with a median survival of 22 weeks. The advantages of the shorter scheme were confirmed.

25-Hydroxyvitamin D3 1alpha-hydroxylase splice variants in breast cell lines MCF-7 and MCF-10.

BACKGROUND: It is known that 25(OH)D3 can be metabolized to 1,25(OH)2 D3 by 1alpha-OHase in breast tissue. This tissue-specific expression of 1alpha-OHase may act as the pivotal link between vitamin D status (25(OH)D3 levels) and the anticancer effects of 1,25(OH)2 D3. Alternative splicing frequently occurs in breast cancer cells; different splice variants of a given protein can display different biological functions and may cause tissue-specific variations. With this study it is the first time that expression and alternative splicing of 1alpha-OHase in the human breast cancer cell line MCF-7 and thebenign breast cell line MCF-10A are described. MATERIALS AND METHODS: Expression of 1alpha-OHase RNA and protein was assessed using a real-time polymerase chain reaction (RT-PCR). The expression of 1alpha-OHase splice variants was detected by a highly specific PCR that combines nested and touchdown PCR. To determine which variants are translated in protein western blot analysis was carried out. RESULTS: The expression of 1alpha-OHase was found to be 1.25-fold higher in MCF-7 compared to MCF-10A cells. In MCF-10A cells, at least 6 splice variants were detected whereas MCF-7 showed no or marginal expression levels of these variants. In MCF-7 cells the antibody detected a signal at 56 kDa corresponding to the size of normal 1alpha-OHase protein. In MCF-10A cells this signal was weaker. In western blot analysis at least two smaller variants at 45 kDa were found in MCF-7 cells. In MCF-10A cells at least 6 proteins between 37 and 56 kDa were detected with an only faint signal. CONCLUSION: We propose that alternative splicing of 1alpha-OHase can regulate the level of active enzyme. Splice variants may lead to a reduction of the protein. The significance of the smaller variants in MCF-7 cells has not been clarified either, but it is known that they are not able to use 25(OH)D3 as a substrate to generate 1,25(OH)D3. In MCF10A cells, more splice variants were identified, it may be that malignant cells contain inactive variants. How far they show a reduced activity remains unclear as no activity measurements were performed.

Metabolism of vitamin D3 in the placental tissue of normal and preeclampsia complicated pregnancies and premature births.

The aim of this study was to analyze the hormonal basis for low 1,25(OH)2D3 circulating levels in patients with preeclampsia and/or preterm deliveries. The activity and expression of the 1 alphaOHase, 25-OHase, 24-OHase and VDR in the placental tissue of normal pregnancies, preeclampsia-complicated pregnancies and premature births were investigated. The mRNA of the enzymes was detected in the placental tissue from preeclamptic pregnancies and compared to those of normal placental tissue. Real time PCR analysis showed a significant increased 1 alpha-OHase gene expression in preeclamptic patients, and the gene expression of 24-OHase was significantly decreased. With regard to the 25-OHase the median value of the normal placental tissue was significantly higher than in the placental tissue of preeclamptic patients. The real time analysis of all target genes also showed significant differences in normal placental tissue compared to placental tissue from premature births (VDR: p = 0.041; 1 alpha-OHase: p = 0.013; 24-OHase: p = 0.007; 25-OHase p = 0.027). Our observation of reduced VDR expression on mRNA level in placental tissue indicates a possible dependence of the modulation of VDR expression from proliferation and differentiation processes. It can be speculated whether the down-regulation of VDR in the examined placenta cells was the result of an altered production of calcitriol by these cells. We found a significantly higher 1 alpha-OHase-expression in the placental tissue of pregnant women with preeclampsia or preterm birth compared to healthy pregnant women, whereas the expression of 25-OHase was significantly reduced. These results correlate with other studies and support the significance of the placenta regarding metabolism malfunctions as they were observed in the calcium metabolism for preeclampsia. That a placenta with preeclampsia expresses less 1 alpha-OHase-mRNA and shows less 1 alpha-OHase-activity than in placental samples of inconspicuous placentae, can be granted as a specific alteration in the placental ability to synthesize adequate amounts of 1,25(OH)2D3.

Expression of splice variants of 1alpha-hydroxylase in mcf-7 breast cancer cells.

1,25-Dihydroxyvitamin D(3) (calcitriol) is the most active natural metabolite of Vitamin D(3). It has strong antiproliferative and differentiating effects on various cell types including breast cancer cells. 25-Hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase, CYP27B1) is one of the key enzymes in the formation of calcitriol. It has been found in breast cancer cells suggesting an autocrine regulation of formation of calcitriol in these cells. Alternative splicing of the encoding genes for this enzyme can possibly play a role in regulating the enzyme level and can explain tissue specific variations of 1alpha-hydroxylase activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. In order to obtain more information on the abundance of 1alpha-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The full-length sequence of 1alpha-hydroxylase and two different splice variants of this enzyme containing intron 1 were isolated. By Western blot technique we then confirmed the protein products of the full-length enzyme and its splice variants. We hypothesize that that the expression of splice variants can lead to a quantitatively lower expression of the mRNA of the full-length enzyme. The abundance of less active 1alpha-hydroxylase protein variants can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues.

Modulation of MAPK ERK1 and ERK2 in VDR-positive and -negative breast cancer cell lines.

1alpha-25-Dihydroxyvitamin D3 (calcitriol), the biologically active metabolite of vitamin D, is known to regulate calcium and phosphate levels in bone metabolism. It is also known to influence proliferation and differentiation in carcinoma cells mediated by the vitamin D receptor (VDR). The antiproliferative effects of calcitriol are believed to be mediated by the nuclear pathway via binding the activated receptor to vitamin D-responsive elements. This induces the vitamin D-responsive genes. Another possible pathway might be the MAPK-cascade or rapid response pathway. The interaction of calcitriol and the MAP-kinase-cascade was evaluated on VDR-positive MCF-7 cells and VDR-negative MDA-MB-231 breast cancer cells. The cells were incubated with calcitriol solution at 10(-7) M and 10(-9) M, or ethanol as controls, for up to 48 h. The effects of calcitriol were measured by semi-quantitative Western blotting. Calcitriol stimulated the MAP-kinases ERK1 and ERK2. A biphasic activation was found for calcitriol in VDR-positive cells after incubation for 5 to 20 min and from 2 to 24 h. However, early activation of ERK1 and ERK2 was also demonstrated in VDR-negative cells. In the controls, ethanol also induced the MAPK-cascade at 5 to 10 min. Calcitriol induction was demonstrated after incubation from 2 to 24 h. In conclusion, it seems that the early induction of the MAPK-cascade was independent of the VDR. A calcitriol-induced MAPK activation was shown after 4 h, which may have been caused by activation of the nuclear receptor pathway.

Endometrial stromal sarcomas--a retrospective analysis of 11 patients.

BACKGROUND: Endometrial stromal sarcoma (ESS) is a malignant tumour with its origin in the endometrial stroma. Little is known about the pathogenesis, risk factors, optimal therapy or outcome of this disease. PATIENTS AND METHODS: Eleven patients with ESS, treated between 1972 and 1996, are reported on. The hospital records of all the patients, including pathology and operative reports, were reviewed and information on treatment, recurrence and survival was obtained. RESULTS: The mean age of our patients was 56.6 years. The main symptom was abnormal vaginal bleeding. Most patients were diagnosed at FIGO stage I. Treatment modalities were surgery, radiation and, in one patient, chemotherapy. The median follow-up time was 42.1 months; 27.3% of the patients had local recurrence. The 1-year, 2-year and 5-year survival rates were 36.3%, 18.1% and 9.1%, respectively. CONCLUSION: ESS is a uterine sarcoma with a difficult differential diagnosis. Patients are frequently diagnosed in an early tumour stage but still experience local or distant recurrence. The prognosis is poor, with early recurrence and low long-time survival rates. The treatment includes surgery and adjuvant radiation, with endocrine therapy being a promising new approach. In order to obtain more information about the pathogenesis of the tumour and to find the optimal therapy, it is necessary that studies, even with small numbers of patients, are undertaken.

[Metastatic breast cancer: options for metastasectomy]. / Das metastasierte Mammakarzinom -- Möglichkeiten einer operativen Therapie.

Usually, the therapy of metastatic breast cancer consists of chemotherapy or endocrine therapy, because even in the case of isolated metastases in one organ, diffuse tumor cell dissemination exists, so that local surgical treatment does not seem sensible. Particurlarly in patients with hepatic or pulmonary metastases the indication for hepatic or pulmonary metastasectomy should be individualized, as hepatic or pulmonary metastases usually develop during a phase of disease, when extrahepatic or -pulmonary metastases also can be detected. Only in patients with long disease-free interval, with isolated hepatic or pulmonary metastases, and the possibility of R0-resection is hepatic or pulmonary metastasectomy a therapeutic option in selected cases.

Right atrial primary cardiac osteosarcoma.

A previously healthy 14-year-old boy with an atypically located primary cardiac osteosarcoma filling the right atrium with significant extension into both systemic and pulmonary venous structures presented with a clinical picture of heart failure. Imaging showed a large right atrial mass with posterior extension into right pulmonary veins and inferior extension into the inferior vena cava causing near total obstruction of systemic venous return to the heart. Bone marrow biopsy showed no evidence of marrow involvement by metastatic malignancy. Histopathology was a high-grade osteosarcoma. Partial tumor debulking was achieved via right atriotomy.

Use of peak Doppler gradient across ventricular septal defects leads to underestimation of right-sided pressures in patients with "sloped" Doppler signals.

In patients with "sloped" appearance of the Doppler signal across a ventricular septal defect (VSD), the peak Doppler velocity seems to overestimate the catheterization-derived peak-to-peak gradient, resulting in underestimation of right-sided heart pressures. In 11 patients with sloped Doppler signals across the VSD, ventricular pressure tracings were compared with simultaneous recordings of the Doppler signal. The average peak Doppler gradient (40.2 +/- 19.2 mm Hg) overestimated the catheterization-derived peak-to-peak gradient (20.2 +/- 13.6 mm Hg) significantly (P < or =.001). Doppler mean gradient (20.2 +/- 11.3 mm Hg; P = ns) and end-systolic gradient (17.0 +/- 12.5 mm Hg; P < or =.05) were closer estimates of the catheterization peak-to-peak gradient. All Doppler gradients showed good correlation to the catheterization peak-to-peak gradient with r2 values of 0.77, 0.73, and 0.91. We conclude that Doppler mean or end-systolic gradients should be used for calculation of right-sided heart pressures in this patient population.

Second-line treatment of fetal supraventricular tachycardia using flecainide acetate.

Digoxin has been an effective treatment for fetal supraventricular tachycardia (SVT), but second-line therapy remains more controversial. Thirty-seven cases of fetal SVT were identified that received digoxin as first-line therapy. Seventeen fetuses (46%) converted to and maintained normal sinus rhythm. Flecainide was used in 13/15 patients requiring second-line therapy; 12/13 (92%) converted to sinus rhythm. Of seven hydropic fetuses, five required second-line therapy and were then successfully converted with flecainide. The improved efficacy of flecainide was statistically significant with a p value <0.01. Complete follow-up was available in 13 digoxin-treated and in 12 second-line therapy infants. Prolonged or multiple drug therapy for postnatal arrhythmia management was required in 3/13 (23%) patients in the digoxin group and in 8/12 (67%) patients requiring second-line therapy. This demonstrated a correlation between the need for second-line fetal therapy and more complex postnatal management with a p value of 0.003. Digoxin remains an effective first-line therapy in the treatment of fetal SVT. Flecainide is an effective second-line therapy, especially in the face of fetal hydrops. Use of second-line therapy in fetal SVT is a predictor of complex postnatal course, and these patients should be followed more closely.

Late onset of pulmonary hypertension after successful Mustard surgery for d-transposition of the great arteries.

Late-onset pulmonary hypertension is a serious complication of Mustard repair for d-transposition of the great arteries. This debilitating complication occurs in 7% of patients who survive to adulthood, even in the face of normal or near-normal postoperative pulmonary pressure.