A természet szénhidrátkémikusai: enzimatikus glikozilezések = Nature's carbohydrate chemists: enzymatic glycosylation

Oligoszacharidok enzimekkel történő előállítása egy igen vonzó alternatív módszer a konvencionális kémiai megközelítés mellett, mert védő csoportok használata nélkül a célvegyület képződését eredményezi. Ezen pályázatban az anomer konfigurációt megtartó alpha amilázok transzglikozilezési képességét tanulmányoztuk humán amiláz szubsztrátok és inhibitorok előállítására. Az akarviozinil-izomaltozil- spiro-tiohidantoin szintézisét a Bacillus stearothermophilus maltogén amilázával (BSMA) oldottuk meg. A BSMA enzim az akarbóz pszeudotriszacharid részét a glüko-spiro-tiohodantoin (GTH) akceptorra transzferálta. A PTS-GTH szerkezetvizsgálata azt mutatta, hogy a glikozilezés főként a glükóz 6-os OH-ján történt az anomer konfiguráció megtartásával. Az akarbózzal meghoszabbított termék több nagyságrenddel hatékonyabb inhibitora a humán amiláznak, mint a kis méretű GTH molekula. A humán amiláz nem rendelkezik transzferáz aktivitással, de mutációval sikerült transzglikozilezési képességet ?bevezetni? a vad típusú enzimbe. A Tyr151Met mutánssal kromoforral jelzett maltooligoszacharidokat állítottunk elő és a PNP 1-tio-béta-maltooligoszacharidokat DP 2-4 izoláltuk. A Tyr151Met mutáns maltotetraóz donorról maltóz és maltotrióz egységeket transzferált különböző PNP glikozidokra. Az NMR analízisek szerint a mutáns enzim megőrízte sztereo-és regioszelektivitását. A glikozilezés a glikozil akceptor 4-es OH-ján történt.Jelenleg árpa amiláz mutánsokkal történő szintézisek folynak. | Enzyme-catalyzed synthesis of oligosaccharides allows the formation of well-defined oligosaccharides selectively without using any protection of hydroxyl groups. In this project the application of transglycosylation ability of retaining glycosidases was studied for the synthesis of oligosaccharide substrates and inhibitors of human amylases. Synthesis of acarviosinyl-isomaltosyl-spiro-thiohydantoin has been achieved by Bacillus stearothermophilus maltogenic amylase (BSMA). BSMA was capable of transferring the acarviosine-glucose residue from an acarbose donor onto glucopyranosylidene-spiro-thiohydantoin (GTH). Structural studies revealed that the enzyme reserved its stereoselectivity. Glycosylation took place mainly at C-6 position. The isolated compound was found to be a much more efficient salivary amylase inhibitor than GTH with kinetic constants of KEI=0.19 uM and KESI= 0.24 uM. Transglycosylation activity has been introduced into human salivary alpha amylase by genetic engineering. Synthesis of PNP 1-thio beta-maltooligosaccharides DP 2-4 has been carried out by a Tyr151Met mutant of HSA. Tyr151Met was capable of transferring maltose and maltotriose residues from a maltotetraose donor onto different PNP glycosides. NMR studies revealed that the mutated enzyme preserved the stereo- and regioselectivity. The glycosylation took place at position 4 of the glycosyl acceptor, exclusively. Nowadays transferase activity of barley amylase mutants has also been studied

Late-night salivary cortisol may be valuable for assessing treatment response in patients with Cushing’s disease: 12-month, Phase III pasireotide study

Measuring salivary cortisol is a simple, convenient and accurate technique with potential value in monitoring patients with hypercortisolism. This analysis reports changes in late-night salivary cortisol (LNSC) during a 12-month, multicentre, Phase III study of patients with Cushing’s disease who were randomized to pasireotide 600 or 900 lg sc bid. LNSC assessment was an exploratory objective based on a single, optional measurement at midnight ± 1 h on the same day as one of the 24-h urinary free cortisol (UFC) measurements. Of 162 enrolled patients, baseline LNSC was measured in 93. Sixty-seven patients had levels above the upper limit of normal (ULN); median baseline levels were 19.7 and 20.7 nmol/L in the groups subsequently randomized to 600 lg (n = 40) and 900 lg (n = 27), respectively. Median LNSC levels decreased from baseline to month 12; median changes in patients who had baseline LNSC [ULN in the 600 and 900 lg groups were -13.4 nmol/L (–52.6 %; n = 19) and -11.8 nmol/L (–56.1 %; n = 14), respectively. LNSC normalized at months 6 and 12 in 25/67 (37.3 %) and 13/67 (19.4 %) patients, respectively; 10/25 and 8/13 patients also had normalized UFC, and 7/25 and 4/13 had partial UFC control (UFC [ULN and C50 % decrease from baseline). There was a moderate correlation (r = 0.55) on the log scale between individual patient LNSC and UFC values when all time points were pooled. Pasireotide decreased LNSC levels during 12 months of treatment. Salivary cortisol may be a simple, convenient biomarker for assessing treatment response in patients with Cushing’s disease

Tissue interactions with lasers and liquid nitrogen: a novel cryopreservation method

The importance of the imposed cooling rate in cryopreserving native cells and tissues has been long recognized in the field of cryobiology. When biological tissues are subjected to cooling rates in excess of thousands of degree C per minute, the characteristic structural and physical manifestations of the ice formed are such that the traditional damage due to ice formation at lower cooling rates are suppressed. Hence, achieving high cooling rates in tissues and cells of biologically relevant sizes (mm’s and cm’s) has been a long standing research problem. In the present study, we present a novel technique to achieve high cooling rates (in the order of 8,000 to 10,000 °C/min) in large tissue sections by coupling pulsed laser heating and immediate exposure to cryogenic temperatures (liquid nitrogen vapor at -164 °C). Thermal gradient that exists between the laser heated tissue (at ~1000’s °C) and liquid nitrogen surrounding the tissue results in very high cooling rates, as opposed to the cooling rates experienced by the tissue without laser heating (which is in the order of a few hundreds of degree C per minute). Furthermore it is expected that the small time scales of energy deposition (6-7 ns) and localized heating due to laser focusing would lead to minimal thermal damage. To illustrate this idea we have developed a 1-D and 2-D numerical model to predict cooling rates experienced in a finite tissue section exposed to liquid nitrogen temperatures with and without laser heating. Based on the numerical results preliminary experiments were carried out in a variety of cryobiologically relevant solutions and using adipose tissue derived adult stem cells. Experimental results indicate the possibility of attaining better survival when cells were cryopreserved using the suggested protocol. The limitations and advantages of the technique are also assessed

P-182: 24-hour ambulatory blood-pressure effects of valsartan + hydrochlorothiazide combinations compared with amlodipine in hypertensive patients at increased cardiovascular risk

In a randomised, double-blind trial, the effects on 24-hr ABP of the combination valsartan 160 mg od and hydrochlorothiazide (HCTZ) 25 or 12.5 mg during 24 weeks of therapy were compared with the effects of amlodipine 10 mg monotherapy (group A10) in 474 stage-II hypertensive patients with additional cardiovascular risk factors. After a two-week single-blind placebo run-in period, patients were randomised to receive valsartan 160 mg od or amlodipine 5 mg od. At Week 4, HCTZ 12.5 mg (group V160/HCTZ12.5) and 25 mg (group V160/HCTZ25) were added to the valsartan groups and in the A10 patients the amlodipine dose was force-titrated to 10 mg od. All treatments reduced BP as well as night-time and daytime BP levels from baseline. 24-hr SBP was reduced by 15.9 ±1.0 mmHg (least-squares mean change ±SE), 19.3 ±1.0 mmHg and 16.1 ±1.1 mmHg in the V160/HCTZ12.5, V160/HCTZ25 and A10 groups, respectively and 24-hr DBP was reduced by 9.3 ±0.6 mmHg, 11.4 ±0.6 mmHg and 9.6 ±0.7 mmHg in the three groups. The differences between the V160/HCTZ25 group and the A10 group were significant (p<0.05) for the changes in 24-hr systolic BP as well as for changes in daytime systolic BP and night-time diastolic BP. Control rates defined as ABPM ≤130/80 mmHg were: 48.4%, 60.8% and 50.9% in the V160/HCTZ12.5, V160/25 and A10 groups, respectively; the differences between the V160/HCTZ25 group and the other two treatment groups were significant at p<0.05. (See Figure) In conclusion, the fixed-dose combination of valsartan 160 mg + HCTZ 25 mg od is an attractive therapeutic option measured on the effects on 24-hr ABPM, night-time and daytime BP reduction and control rates in hypertensive patients at additional cardiovascular ris

Kutatások a MALDI-TOF tömegspektrometria szénhidrátkémia alkalmazási lehetőségeinek kiszélesítésére = Investigation of widening of the MALDI-TOF MS applications in carbohydrate chemistry

A kutatás fő célkitűzése volt szénhidrátok és származékaik (védőcsoportokat tartalmazó vagy fehérjéhez kötött oligoszacharidok) MALDI-TOF tömegspektrometriás meghatározásához optimális kísérleti körülmények megállapítása és így a szénhidrátkémiai kutatások segítése. Ciklodextrin modellvegyületek alkalmazásával vizsgáltuk a minta előkészítés és felvitel valamint a kísérleti paraméterek változtatásának hatását a spektrumra. Tanulmányozzuk a mátrix anyagának, a minta koncentrációjának, a mintafelvitel módjának és a reflektron detektor érzékenység hatását a spektrum minőségére. Védőcsoportokat tartalmazó oligoszacharidok mérésekor számos törvényszerűséget figyeltünk meg a védőcsoportok stabilitása, a különböző mátrixok és kísérleti paraméterek között. A MALDI mérések segítették a termékek és melléktermékek azonosítását kémiai és enzimes oligoszacharid szintézisek esetében. Fragmentációs vizsgálatokkal kémiai és enzimatikus szintézissel előállított oligoszacharidok, valamint természetes eredetű amiláz inhibítorok szerkezet felderítését végeztük el. Meghatároztuk különböző neoglikoproteinek oligoszacharid tartalmát, amely meghatározó a sikeres immunválasz kiváltásában. Méréseinkkel támogattuk a különböző neoglikoprotein előállítási módszerek szisztematikus vizsgálatát. | The object of this research program was the application of MALDI-TOF MS in the field of carbohydrates and glycoconjugates. Cyclodextrins as model compounds were used to investigate the effect of the sample preparation methods and experimental parameters on the spectrum. The studied experimental parameters were: different matrix compounds, detector sensitivity, sample concentration and sample preparation methods. The stability of the protecting groups under the conditions of the evaporation/ionization was studied. The MALDI measurements assisted to identify products and byproducts of different chemical and enzymatic oligosaccharide syntheses (eg. sugar-sulfonates, sulfonic acid esters, PNP-glycosides and acarbose derivatives). Structural analysis and verification were made using post source decay fragmentation method in case of a tetrasaccharid component of N-glycanes, acarbose and its derivatives obtained by enzymatic synthesis and some natural alpha-amylase inhibitors. In addition we used this method for determination of molecular masses of neoglycoproteins

Unveiling Identity: Exploring Afrofuturism in Ekow Nimako’s Contemporary African Diasporic Sculptural Art

Identity expressed within African diasporic arts has historically been connected to traditional genres such as portraiture. Over time, contemporary artists have explored identity through genres beyond portraiture and through the use of non-traditional materials. The sculptural practice of Ghanaian Canadian artist Ekow Nimako, a fine arts sculptor based in Toronto, Canada, employs the unconventional material of LEGO® to offer a multi-generational perspective into deep diasporic memory. Examining Nimako’s sculptures through the perspective of colonialism and de-colonialism, materiality, and Afrofuturism, this thesis investigates the artist’s exploration of Black historical pasts to shape identities and construct narratives of Black futures. The monumental sculpture Kumbi Saleh 3020 CE emerges as a nuanced exploration of identity intricately connected to responses to colonial legacies. Nimako utilizes speculative storytelling to challenge colonial historical records by envisioning a future where Black individuals actively shape their own narratives. Nimako’s specific use of black LEGO® material enriches his works and underscores the significance of Black identity in historical and future contexts. Kumbi Saleh 3020 CE becomes a tangible exploration of identity, challenging prevailing stereotypes about Africa and Africans of the diaspora and presenting a futuristic city where Black agency in identity formation is unshackled from colonial constraints. Engaging themes such as feminism, resistance to oppression, and the reimagining of highly technologically advanced Black individuals, Nimako’s sculptural narratives surpass mere assertions of Black presence. They vividly illustrate the forward reaching results of Black agency, resilience, and innovation, and they make a substantial contribution to the ongoing discourse on identity within Afrofuturism. This work represents a unique analysis that contributes substantially to the art historical discourse on identity, contemporary art, Afrofuturism, and the sculptural practice of Ekow Nimako