High-resolution imaging reveals a limit in spatial resolution of blood flow measurements by microspheres

Density of 15-microm microspheres after left atrial application is the standard measure of regional perfusion. In the heart, substantial differences in microsphere density are seen at spatial resolutions <5 ml, implying perfusion heterogeneity. Microsphere deposition imaging permits a superior evaluation of the distribution pattern. Therefore, fluorescent microspheres (FMS) were applied, FMS deposition in the canine heart was imaged by epifluorescence microscopy in vitro, and the patterns were observed compared with MR images of iron oxide microspheres (IMS) obtained in vivo and in vitro. FMS deposition in myocardial slices revealed the following: 1) a nonrandom distribution, with sequentially applied FMS of different color stacked within the same vessel, 2) general FMS clustering, and 3) rather large areas devoid of FMS (n = 3). This pattern was also seen in reconstructed three-dimensional images (<1 nl resolution) of FMS distribution (n = 4). Surprisingly, the deposition pattern of sequentially applied FMS remained virtually identical over 3 days. Augmenting flow by intracoronary adenosine (>2 microM) enhanced local microsphere density, but did not alter the deposition pattern (n = 3). The nonrandom, temporally stable pattern was quantitatively confirmed by a three-dimensional intermicrosphere distance analysis of sequentially applied FMS. T2-weighted short-axis MR images (2-microl resolution) of IMS revealed similar patterns in vivo and in vitro (n = 6), as seen with FMS. The observed temporally stable microsphere patterns are not consistent with the notion that microsphere deposition is solely governed by blood flow. We propose that at high spatial resolution (<2 microl) structural aspects of the vascular network dominate microsphere distribution, resulting in the organized patterns observed.     

Distribution of Chitinases in the Entomopathogen <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Effect of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine in Protein Secretion

For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.     

Enhancement of vitamin E production in sunflower cell cultures

The most biologically active component of vitamin E, -tocopherol, is synthesized in its most effective stereoisomeric form only by photosynthetic organisms. Using sunflower cell cultures, a suitable in vitro production system of natural -tocopherol was established. The most efficient medium was found to be MS basal medium with naphthaleneacetic acid and 6-benzylaminopurine with the addition of casaminoacids and myo-inositol. Culture feeding experiments using biosynthetic precursors showed that -tocopherol production improved by 30% when homogentisic acid was used. Interestingly, time-course experiments with sunflower suspension cultures showed a possible increase of 78% in -tocopherol production when using cultures of longer subculture intervals. Compared to the starting plant tissue, an overall 100% increase of -tocopherol was reached by these sunflower cell cultures.     

AmSUT1, a sucrose transporter in collection and transport phloem of the putative symplastic phloem loader Alonsoa meridionalis

A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80-91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.     

Bipolar anastomosis technique with removable instruments: an easy, fast, and reliable technique for vascular anastomosis

The interrupted suture technique is most commonly used for microsurgical vascular anastomosis. For several reasons (e.g., exposure of suture material to blood, time needed), many attempts have been made to find other solutions. This article describes a new means of performing a microsurgical vascular anastomosis. The aim of this study was to show the feasibility and possible advantages of this new technique. The basic components at work here are a modified cuff and electrically generated heat used to unite the vessel walls. In this way, both endothelial layers are adapted without manipulating the inside of the vessel or leaving behind foreign matter. Various energy/coagulation time settings were used to perform arterial anastomoses (n = 42) in an isogeneic abdominal aorta interposition model in the rat. The quality of anastomosis was evaluated at days 1, 10, 21, and 120. Immediately after the welding process all anastomoses (n = 42) were patent. No stenosis was found at any observation time. Anastomosis time ranged from 3 to 18 minutes (average, 11 minutes). This new technique permits a vascular anastomosis to be performed easily and reliably with a high patency rate. With this technique, the authors are convinced that a skilled surgeon can create a high-quality anastomosis in a fraction of the time needed to sew an anastomosis.     

Use of dermal regeneration template in contracture release procedures: a multicenter evaluation

Integra dermal regeneration template (Integra Life Sciences, Plainsboro, N.J.) is an effective treatment for full-thickness burns. It can also be useful in contracture release procedures; however, the clinical utility of a dermal regeneration template in contracture release procedures has not been adequately characterized. In this multicenter investigation, the outcomes of release procedures incorporating a dermal regeneration template for 89 consecutive patients, who underwent a total of 127 contracture releases, were retrospectively evaluated. The procedures involved the application of Integra, which includes a temporary silicone epidermal substitute and an artificial dermal layer. After formation of a neodermis, the silicone layer is removed and replaced with an epidermal autograft. Data on patient and contracture site history, treatment methods, physician assessments of range of motion or function, patient satisfaction, recurrence, and adverse events were collected with a standardized questionnaire. Release procedures for the study patients involved the neck, axilla, trunk, elbow, knee, hand, and other anatomical sites. The mean postoperative follow-up period was 11.4 months. At 76 percent of the release sites, range of motion or function was rated as good (significant improvement in range of motion or function) or excellent (maximal range of motion or function possible) by physicians. Responding patients expressed satisfaction with the overall results of treatment at 82 percent of the sites. No recurrence of contracture at 75 percent of the sites was observed during follow-up monitoring. Patient age and prior surgical treatment at the site did not significantly affect the results of treatment. However, outcomes were superior at mature sites, i.e., those for which more than 12 months had elapsed since the original injury. Postoperative complications rarely necessitated regrafting. These results indicate that a dermal regeneration template provides a useful alternative technique for contracture release procedures. The study data indicate that this approach leads to favorable functional outcomes and a high rate of patient satisfaction. This modality also seems to be versatile, because a range of anatomical sites are amenable to treatment with a dermal regeneration template, regardless of prior surgical treatment, and both pediatric and adult patients respond well to this form of therapy. Furthermore, Integra confers functional and cosmetic benefits similar to those of full-thickness grafts but without comparable potential for donor-site morbidity.     

The effects of mixotrophy on the stability and dynamics of a simple planktonic food web model

Recognition of the microbial loop as an important part of aquatic ecosystems disrupted the notion of simple linear food chains. However, current research suggests that even the microbial loop paradigm is a gross simplification of microbial interactions due to the presence of mixotrophs-organisms that both photosynthesize and graze. We present a simple food web model with four trophic species, three of them arranged in a food chain (nutrients-autotrophs-herbivores) and the fourth as a mixotroph with links to both the nutrients and the autotrophs. This model is used to study the general implications of inclusion of the mixotrophic link in microbial food webs and the specific predictions for a parameterization that describes open ocean mixed layer plankton dynamics. The analysis indicates that the system parameters reside in a region of the parameter space where the dynamics converge to a stable equilibrium rather than displaying periodic or chaotic solutions. However, convergence requires weeks to months, suggesting that the system would never reach equilibrium in the ocean due to alteration of the physical forcing regime. Most importantly, the mixotrophic grazing link seems to stabilize the system in this region of the parameter space, particularly when nutrient recycling feedback loops are included.     

Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural studies of the 1,3-dichloropropene catabolic enzymes

The use of the soil fumigant Telone II, which contains a mixture of cis- and trans-1,3-dichloropropene, to control plant-parasitic nematodes is a common agricultural practice for maximizing yields of various crops. The effectiveness of Telone II is limited by the rapid turnover of the dichloropropenes in the soil due to the presence of bacterial catabolic pathways, which may be of recent origin. The characterization of three enzymes in these pathways, trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD), has uncovered intriguing catalytic mechanisms as well as a fascinating evolutionary lineage for these proteins. Sequence comparisons and mutagenesis studies revealed that all three enzymes belong to the tautomerase superfamily. Tautomerase superfamily members with known structures are characterized by a β-α-β structural fold. Moreover, they have a conserved N-terminal proline, which plays an important catalytic role. Mechanistic, NMR, and pH rate studies of the two dehalogenases, coupled with a crystal structure of CaaD inactivated by 3-bromopropiolate, indicate that they use a general acid/base mechanism to catalyze the conversion of their respective isomer of 3-chloroacrylate to malonate semialdehyde. The reaction is initiated by the conjugate addition of water to the C-2, C-3 double bond and is followed by the loss of HCl. MSAD processes malonate semialdehyde to acetaldehyde, and is the first identified decarboxylase in the tautomerase superfamily. The catalytic mechanism is not well defined but the N-terminal proline plays a prominent role and may function as a general acid catalyst, similar to its role in CaaD and cis-CaaD. These are the first structural and mechanistic details for tautomerase superfamily members that catalyze either a hydration or a decarboxylation reaction, rather than a tautomerization reaction, in which Pro-1 serves as a general acid catalyst rather than as a general base catalyst. The available information on the 1,3-dichloropropene catabolic enzymes allows speculation on the possible evolutionary origins of their activities.