The chemistry and use of cellulose derivatives for the study of biological systems

Cellulose chemistry and applications as carrier for proteins, polymers, and organic molecule

NOX Inhibitors - A Promising Avenue for Ischemic Stroke.

NADPH-oxidase (NOX) mediated superoxide originally found on leukocytes, but now recognized in several types of cells in the brain. It has been shown to play an important role in the progression of stroke and related cerebrovascular disease. NOX is a multisubunit complex consisting of 2 membrane-associated and 4 cytosolic subunits. NOX activation occurs when cytosolic subunits translocate to the membrane, leading to transport electrons to oxygen, thus producing superoxide. Superoxide produced by NOX is thought to function in long-term potentiation and intercellular signaling, but excessive production is damaging and has been implicated to play an important role in the progression of ischemic brain. Thus, inhibition of NOX activity may prove to be a promising treatment for ischemic brain as well as an adjunctive agent to prevent its secondary complications. There is mounting evidence that NOX inhibition in the ischemic brain is neuroprotective, and targeting NOX in circulating immune cells will also improve outcome. This review will focus on therapeutic effects of NOX assembly inhibitors in brain ischemia and stroke. However, the lack of specificity and toxicities of existing inhibitors are clear hurdles that will need to be overcome before this class of compounds could be translated clinically

Prorenin and the heart : the Mannose 6-phosphate connection

The knowledge concerning the formation of angiotensins at cardiac tissue sites in relation to the presence and origin of cardiac renin, angiotensinogen and ACE is evaluated in chapter 2. To gain insight in the functional importance of locally generated angiotensin 11, the response of human forearm blood flow to infusion of either angiotensin I or angiotensin 11 was investigated (Chapter 3). To extend our results in the perfused isolated rat heart,31 experiments were performed to detect de novo synthesis of RAS components by neonatal rat cardiomyocytes and -fibroblasts under basal conditions and after stretch (Chapter 4). In addition, we characterized the binding and activation of human recombinant prorenin via mannose 6- phosphate/IGF11 receptors on the surface of human endothelial cells, and neonatal rat cardiomyocytes and -fibroblasts (Chapters 5 and 6). To validate our results obtained with human recombinant prorenin, neonatal rat cardiomyocytes were also incubated with human (pro)renin- containing body fluids (Chapter 7). The latter studies also addressed the importance of soluble mannose 6-phosphate/IGF11 receptors. Finally, since 1) under certain conditions man nose 6-phosphate/IGF11 receptor activation initiates transcellu\ar signaling pathways,'2 and 2) renin binding to glomerular mesangial cells leads to plasminogen activator inhibitor type-1 release and an increase in 3H-thymidine incorporation,25 we investigated whether prorenin binding and/or uptake by rat cardiomyocytes, in the presence or absence of angiotensinogen, resulted in a cellular response (Chapter 8). In these latter studies we also investigated intra- and extracellular angiotensin 11 generation and compared the effects of prorenin with those obtained with angiotensin II in parallel experiments

High impact

Journal ArticleFrom 1993-97, Gilbert contributed to seven articles published in top peer-reviewed journals, which were then cited 1,157 times, or an average of 165.3 times per paper. When Gilbert heard the news, "it kind of blew me away." He acknowledged that the articles, clinical trials of new drugs to treat chronic heart failure, "are pivotal findings. " As gratifying as the accolades are, Gilbert is proudest of the clinical impact: "Patients now are getting these drugs prescribed because of these papers." About 5 million Americans have chronic heart failure, sometimes referred to as congestive heart failure. Chronic heart failure usually is a progressive condition. Chronic heart failure is on the rise. "It's the most common cause of hospitalization in the elderly," said Gilbert. These hospitalizations cost a total of $8-15 million each year, more than twice the cost of hospitalizations for all forms of cancer. Fortunately, as reported in the high-impact papers, angiotensin-converting enzyme (ACE) inhibitors and beta-adrenergic receptor (beta) blockers not only treat the symptoms of chronic heart failure, but also reverse the progression. "Taken together, these drugs will reduce the annual rate of death from heart failure by 50 percent," said Gilbert

Angiotensin-I-Converting Enzyme and its Relatives

Angiotensin-I-converting enzyme (ACE) is a monomeric, membrane-bound, zinc- and chloridedependent peptidyl dipeptidase that catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II, by removing a carboxy-terminal dipeptide. ACE has long been known to be a key part of the renin angiotensin system that regulates blood pressure, and ACE inhibitors are important for the treatment of hypertension. There are two forms of the enzyme in humans, the ubiquitous somatic ACE and the sperm-specific germinal ACE, both encoded by the same gene through transcription from alternative promoters. Somatic ACE has two tandem active sites with distinct catalytic properties, whereas germinal ACE, the function of which is largely unknown, has just a single active site. Recently, an ACE homolog, ACE2, has been identified in humans that differs from ACE in being a carboxypeptidase that preferentially removes carboxy-terminal hydrophobic or basic amino acids; it appears to be important in cardiac function. ACE homologs (also known as members of the M2 gluzincin family) have been found in a wide variety of species, even in those that neither have a cardiovascular system nor synthesize angiotensin. X-ray structures of a truncated, deglycosylated form of germinal ACE and a related enzyme from Drosophila have been reported, and these show that the active site is deep within a central cavity. Structure-based drug design targeting the individual active sites of somatic ACE may lead to a new generation of ACE inhibitors, with fewer side-effects than currently available inhibitors

Identification of Retinoic Acid in a High Content Screen for Agents that Overcome the Anti-Myogenic Effect of TGF-Beta-1

Transforming growth factor beta 1 (TGF-β1) is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. When neutralizing antibodies to TGF-β1 or the angiotensin II inhibitor losartan were used to reduce TGF-β1 signaling, muscle morphology and function were restored in mouse models of Marfan Syndrome and muscular dystrophy. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-β1.A high-content cell-based assay was developed in a 96-well plate format that detects the expression of myosin heavy chain (MHC) in C2C12 cells. The assay was used to quantify the dose-dependent responses of C2C12 cell differentiation to TGF-β1 and to the TGF-β1 Type 1 receptor kinase inhibitor, SB431542. Thirteen agents previously described as promoting C2C12 differentiation in the absence of TGF-β1 were screened in the presence of TGF-β1. Only all-trans retinoic acid and 9-cis retinoic acid allowed a maximal level of C2C12 cell differentiation in the presence of TGF-β1; the angiotensin-converting enzyme inhibitor captopril and 10 nM estrogen provided partial rescue. Vitamin D was a potent inhibitor of retinoic acid-induced myogenesis in the presence of TGF-β1. TGF-β1 inhibits myoblast differentiation through activation of Smad3; however, retinoic acid did not inhibit TGF-β1-induced activation of a Smad3-dependent reporter gene in C2C12 cells.Retinoic acid alleviated the anti-myogenic effect of TGF-β1 by a Smad3-independent mechanism. With regard to the goal of improving muscle regeneration and function in individuals with muscle disease, the identification of retinoic acid is intriguing in that some retinoids are already approved for human therapy. However, retinoids also have well-described adverse effects. The quantitative, high-content assay will be useful to screen for less-toxic retinoids or combinations of agents that promote myoblast differentiation in the presence of TGF-β1

Enantiomeri kinuklidin-3-ol derivata: Razdvajanje enantiomera i interakcija s ljudskim kolinesterazama

The (R)- and (S)-enantiomers of quinuclidin-3-ol and quinuclidin-3-yl acetate as well as their quaternary N-methyl and N-benzyl derivatives were synthesized in order to study the stereoselectivity of human erythrocyte acetylcholinesterase (EC 3.1.1.7) and plasma butyrylcholinesterase (EC 3.1.1.8). The compounds were tested as substrates and inhibitors of cholinesterases. Both cholinesterases hydrolyze the derivatives of quinuclidin-3-yl acetate with a preference for the (R)- over (S)-enantiomers. In contrast to the hydrolysis of the enantiomers of acetates, the inhibition of acetylcholinesterase and butyrylcholinesterase by the (R)- and (S)-enantiomers of quinuclidin-3-ol derivatives does not reveal enantiomeric preference of the enzymes. The (R)- and (S)-acetates also act as nonstereoselective inhibitors of the enzyme-induced hydrolysis of acetylthiocholine. The best substrate is (R)-N-methyl-3-acetoxyquinuclidinium iodide with kcat = 1.5 x 106 min–1 and kcat = 5.5 x 104 min–1 for acetylcholinesterase and butyrylcholinesterase, respectively. The (R)- and (S)-N-benzylquinuclidinium derivatives are the most potent inhibitors of both enzymes.Priređeni su (R)- i (S)-enantiomeri kinuklidin-3-ola i kinuklidin-3-il-acetata te odgovarajući kvaterni N-metilni i N-benzilni derivati kako bi se proučila njihova interakcija s ljudskom eritrocitnom acetilkolinesterazom (EC 3.1.1.7) i butirilkolinesterazom iz plazme (EC 3.1.1.8). Spojevi su studirani kao supstrati i inhibitori tih enzima. Obje kolinesteraze pokazuju visoku stereoselektivnost pri hidrolizi kinuklidin-3-il acetata preferirajući (R)- u odnosu na (S)-enantiomere. Nasuprot hidrolizi enantiomera acetatnih derivata, inhibicija acetilkolinesteraze i butirilkolinesteraze s (R)- i (S)-enantiomerima kinuklidin-3-ola i kinuklidin-3-il-acetata te njihovih N-metilnih i N-benzilnih derivata, ne pokazuje stereoselektivnost tih enzima. Kao najbolji supstrat za oba enzima pokazao se (R)-N-metilkinuklidinijev acetat, s kcat = 1,5 x 106 min–1 za acetilkolinesterazu, odnosno kcat = 5,5 x 104 min–1 za butirilkolinesterazu. (R)- i (S)-N-benzilkinuklidinijevi derivati bili su najjači inhibitori za te enzime

The use of cardiac biomarkers in veterinary medicine: the equine perspective

In human medicine, cardiac biomarkers, such as natriuretic peptides and troponins, are routinely used for the diagnosis, prognosis and monitoring of heart diseases. Similarly, these biomarkers are determined in small animals to differentiate non-cardiac from cardiac diseases. Knowledge about these biomarkers in horses is limited and requires further investigation. The first equine studies about atrial natriuretic peptide (ANP) and N-terminal ANP (NT-proANP) are promising, and show a clear correlation with atrial dimension size. Equine brain natriuretic peptides assays are still unavailable. The troponins, in particular troponin I, have been more extensively studied in horses, and their use for the diagnosis of myocardial damage has been fully demonstrated. They have replaced the less specific lactate dehydrogenase and creatine kinase isoenzymes, which makes the use of the last mentioned no longer legitimate. A final possible equine biomarker is aldosterone. Reference values in horses have been established. However, in only one study, a correlation between aldosterone and cardiac disease has been reported