Electroporation of the vasculature and the lung

Electroporation has proven to be a highly effective technique for the in vivo delivery of genes to a number of solid tissues. In most of the reported methods, DNA is injected into the target tissue and electrodes are placed directly on or in the tissue for application of the electric field. While this works well for solid tissues, there are many tissues and organs that are not amenable to such an approach. In this review I will focus on the development of electroporation protocols for two such tissues: the vasculature and the lung. Several methods for in vivo electroporation of the vasculature have been developed in recent years that deliver DNA to vessel segments from either the inside or outside of the vessel. The advantages and disadvantages of each are discussed, as are the applications for which they have been used. In more recent work, our laboratory has developed a novel method to deliver genes to the rodent lung that results in high level, uniform, gene expression throughout all cell types of the lung. Most importantly, this technique is safe, and causes no inflammatory response or alterations in normal physiology of the organs. Taken together, these studies demonstrate the utility of electroporation for gene transfer to non injectible tissues.     

Maximizing the use of the abdominoplasty incision

Candidates for abdominoplasty often request multiple procedures at the time of consultation. Some of these patients have the potential opportunity to have ancillary procedures performed through the abdominoplasty incision, such as breast augmentation or suction-assisted lipectomy. Access via the abdominoplasty incision can also limit the need for distant donor sites, for instance, when autologous fillers or rib graft are necessary. The techniques described are straightforward and are based on standard principles that should be considered when ancillary procedures are performed in conjunction with abdominal contouring procedures. In a review of 70 consecutive patients undergoing abdominoplasty, 91 ancillary procedures were performed in conjunction with the abdominoplasty. Of the total number of patients undergoing abdominoplasty, 29 patients underwent 30 procedures facilitated through their abdominoplasty incision, including 23 suction-assisted lipectomies of the flanks, six breast augmentations, and one rib cartilage harvesting for rhinoplasty. A review of the author's experience and discussion for potential options afforded by this exposure are presented.     

Distribution and properties of the genes encoding the biosynthesis of the bacterial cofactor, pyrroloquinoline quinone

Pyrroloquinoline quinone (PQQ) is a small, redox active molecule that serves as a cofactor for several bacterial dehydrogenases, introducing pathways for carbon utilization that confer a growth advantage. Early studies had implicated a ribosomally translated peptide as the substrate for PQQ production. This study presents a sequence- and structure-based analysis of the components of the pqq operon. We find the necessary components for PQQ production are present in 126 prokaryotes, most of which are Gram-negative and a number of which are pathogens. A total of five gene products, PqqA, PqqB, PqqC, PqqD, and PqqE, are identified as being obligatory for PQQ production. Three of the gene products in the pqq operon, PqqB, PqqC, and PqqE, are members of large protein superfamilies. By combining evolutionary conservation patterns with information from three-dimensional structures, we are able to differentiate the gene products involved in PQQ biosynthesis from those with divergent functions. The observed persistence of a conserved gene order within analyzed operons strongly suggests a role for protein-protein interactions in the course of cofactor biosynthesis. These studies propose previously unidentified roles for several of the gene products, as well as identifying possible new targets for antibiotic design and application.     

On the interpretation of the effect of area on the critical flicker frequency

The effects of area and intensity on the critical flicker frequency, threshold, and reaction time are considered in terms of neural net theory. An attempt is made to develop a mechanism which can account for the phenomena associated with the empirically observed laws of Ricco, Granit, Talbot, and Ferry-Porter as well as observations on reaction time and threshold. A simple model gives results which are substantially in agreement with observation except for a few apparent discrepancies. Experimental procedures are suggested which can determine whether these are apparent or real. This research was supported by the United States Air Force through the Air Force Office of Scientific Research of the Air Research and Development Command under contract No. AF 18(600)-1454. Reproduction in whole or in part is permitted for any purpose of the United States Government.     

Perspectives of the control of Opisthorchis invasion by the method of treating the molluskan population]

Sanitation of the mollusk population with highly effective cercaricide preparations can be used for the control of opisthorchosous infection. Low concentrations of molluskocides which are sublethal for non-infected mollusks have turned to be lethal for hyperinfected individuals. All tested molluskocides display a cercaricide effect in concentrations which are considerably lower than molluskocide ones. Low concentrations of cercaricides are toxic both for cercariae and parthenites from the liver of mollusks and for freely swimming cercariae. Disastrously affecting the freely swimming cercariae microdoses of cercaricides prevent the infection of the second intermediate hosts. Microdoses of cercaricides which are lethal for cercariae are not toxic for the majority of hydrobionts including fishes of all age groups. As far as the dynamics of the infection level of Bithynia with O. felineus is characterized by a one-peak curve with a distinct peak in the middle-end of July the treatment of water bodies with microdoses of cercaricides should be carried out just in this period.     

Innervation of the anococcygeus muscle of the rat

Summary The innervation of the anococcygeus muscle of the rat was investigated with regard to the histochemical features of nerve fibers within the muscle and to the location of the postganglionic autonomic neurons which are the source of these fibers. Acetylcholinesterase-positive fibers and catecholaminergic fibers are abundant in the anococcygeus as well as the related retractor penis muscle. Neuronal somata, either between muscle bundles of the anococcygeus or in the connective tissue sheath, are also acetylcholinesterase-positive. Nerve fibers and a minority of the ganglion cells in the anococcygeus and retractor penis muscles are immunoreactive for vasoactive intestinal polypeptide. Injection of the retrogradely transported dye Fluorogold into the anococcygeus muscle filled neurons in the abdominopelvic sympathetic chain, pelvic plexus and a small number of neurons in the inferior mesenteric ganglion. In the pelvic plexus, some neurons were located in the major pelvic ganglion but most were found along the main penile nerve and its branches to the anococcygeus muscle. Immunocytochemistry of these identified neurons indicates that about one half of them are positive for vasoactive intestinal polypeptice. These results raise the possibility that both acetylcholine and vasoactive intestinal polypeptide are important neurotransmitters in autonomic nerves to the anococcygeus muscle.     

A mesoscopic technique for the study of the development of the peripheral system in rat foetuses

In order to study the morphogenesis of the nervous system in the rat an acetylcholinesterase in toto method for staining nervous tissue in rat foetuses was developed. Procedure: Rat foetuses of 14-22 days are fixed "en bloc" for 24 hours in a cold sucrose-formol solution. Fixed specimens are rinsed for 2 days in cold 0.22 M sucrose in a sodiumcacodylate buffer (pH 7.2). The specimens are cut (mid-)sagittally with the aid of a razor-blade, and incubated in a medium of acetylthiocholine iodide in acetate buffer (pH 5.0). Then, dehydration in glycerine/water mixtures of increasing glycerine content follows. The specimens may be stored in pure glycerine or embedded in epoxy-resin blocks and can be studied under a binocular dissecting microscope. In using this in toto staining method both the continuity of the central and peripheral parts of the nervous system as well as details up to the level of individual perikarya and motor endplates are preserved. With this mesoscopic method the three-dimensional architecture of the peripheral nervous system and its topological relations to other structures can be studies in one specimen. The exact procedure and the results as well as a method for embedding specimens in epoxy-resin blocks for teaching purposes are described. The advantages of this mesoscopic staining method for foetuses are discussed.     

Quantitative analysis of the structure of the varicose dilatations in the nerve fibers of the rabbit coronary artery (according to the results of three-dimensional reconstruction)]

Axoplasmic vesicles and microtubes in varicosities of axonal plexus in the external sheath of the rabbit coronary artery have been studied. Comparing serial sections and examining three-dimensional reconstruction of small nerve plexus, it was demonstrated that various varicosities differed only in their correlation of the amount of small (30-80 nm) and large (80-180 nm) vesicles. Average diameters in profiles of small and large vesicles are 56.3 nm and 115.6 nm, respectively. There are varicosities containing about 250 or more than 1,000 vesicles. Evenly distributed vesicles throughout the volume of varicosities and lack of specialized structures on the axolemma are supposed to demonstrate the absence of special areas for the mediator removal in the axons studied. The microtubes in the varicosities are peripherally arranged, next to the axolemma and form 1-1.5 wide coils. A suggestion is made that the varicosities in neighbouring axons of the same nerve plexus, with specific structural organization, are special functional units and appear to be peculiar not only for nerve plexus of the coronary artery, but also for other parts of the peripheral vegetative nerve system.     

Comparative studies on the mechanism of activation of the two human trypsinogens

The activation of human trypsinogens 1 and 2 by porcine enterokinase at pH 5.6 shows that the two human zymogens are equivalent substrates for this enzyme and that both proteins are activated faster than the cationic bovine trypsinogen. At pH 8.0 and in the presence of 20 mM calcium the two human trypsinogens are activated by either human trypsin at the same rate but the affinity of both trypsins is higher for trypsinogen 1 than for trypsinogen 2. Two Ca2+ binding sites are identified in the two human zymogens and their pK(Ca2+) values determined. For trypsinogen 1 the values are respectively of 2.8 and 3.3 for the primary and secondary Ca2+ binding sites, and for trypsinogen 2 of 3.4 and 2.7. These values are markedly different from those obtained for bovine cationic trypsinogen, especially in the case of trypsinogen 1. These results point out a different degree of saturation of the calcium binding sites of the 2 human zymogens that must exist in physiological conditions, suggesting different biological activities of the two trypsinogens.     

Applications of the Mantel-Haenszel statistic to the comparison of survival distributions.

In comparing two survival distributions, a Mantel-Haenszel statistic can be computed after each death as a non-linear two-sample rank statistic. The distributions of both the maximum and terminal statistics in such a sequence are studied numerically, in the absence of censoring, and appropriate critical values are determined. The maximum statistic is applied to simultaneous inference, and both the maximum and terminal statistics are used as the basis for early stopping procedures (especially in the pseudo-sequential context). Procedures based on the two statistics are compared for power and for early decision properties such as stopping index and (for exponential distributions) stopping time.     

Model for the conformational analysis of hydrated peptides. Effect of hydration on the conformational stability of the terminally blocked residues of the 20 naturally occurring amino acids

The effects of hydration are included in empirical conformational energy computations on oligopeptides by means of a modified hydration-shell model. Free energy terms are introduced to account for “specific hydration” due to water–solute hydrogen bonding and for “nonspecific hydration” describing the interaction of the solute with water molecules in a first-neighbor shell. The dielectric constant has been doubled (over the value used for calculations in the absence of water) to take into account the presence of solvent. Computations were carried out for the N-acetyl-N′-methylamides of the 20 naturally occurring amino acids. Conformational energy maps are compared with similar maps calculated in the absence of hydration. Minimum-energy conformations are located and compared with the corresponding minima for unhydrated peptides in terms of ordering with respect to potential energy, the dihedral angles at the minima, and the presence of intramolecular hydrogen bonds. The Boltzmann factors for various conformational regions are altered significantly on hydration in some cases. These changes can be explained in terms of differences in the hydration free energy terms for various conformations.     

Perspectives of the conservation of the marine and coastal ecosystems in the frame of the realization of the Convention on Biological Diversity

The specifics of the international and regional activity in the context of the realization of the Convention on Biological Diversity has been analyzed. The global aims, tasks, and criteria of the working program of the Convention on marine and coastal biodiversity are discussed. The actual direction of the investigations is analyzed; program actions are planned; arrangements essential for conservation of the biodiversity of marine and coastal ecosystems are proposed.     

Some features of the ultrastructure of the coenecium of Rhabdopleura compacta

Summary The coenecium of Rhabdopleura consists of a series of tubes, some erect and some repent. These tubes are composed of rings, one stacked within another. The rings are smooth on the inside surface and rough outside. Newly laid down rings are thin and smooth on both surfaces, fibrous material is laid down on the external surface during growth in thickness by the cephalic shield of the zooid. The erect tubes remain discrete, but the repent tubes, which are attached to the substratum can become incorporated in a mass of secreted material. The external vertical fibres cross several rings and probably serve to anchor the stack. Besides these fibrils that run for several segments, there are other shorter fibres that run along the length of each cylindrical ring, and are not continuous across the rings. These long and short fibres have features in common with those found in the graptolites.I wish to thank Dr. A. Boyde for scanning electron microscope facilities and for making his expertise so freely available. Dr. A. Stebbing helped me to obtain the specimens. Mrs. E. Bailey and Mr. R. Moss ably provided the technical and photographic assistance     

Taking the good out of the bad: lentiviral-based gene therapy of the hemoglobinopathies

Sickle cell disease and beta-thalassemia are excellent candidates for gene therapy since transfer of a single gene into hematopoietic stem cells should theoretically elicit a therapeutic response. Initial attempts at gene therapy of these hemoglobinopathies have proved unsuccessful due to limitations of available gene transfer vectors. With the extensive research on human immunodeficiency virus-1 due to the acquired immune deficiency syndrome pandemic, researchers have realized that this lentivirus, engineered to be devoid of any pathogenic elements, can be an effective gene transfer vector. This review discusses the gene therapy strategy for the hemoglobinopathies and outlines why lentiviral-derived vectors are particularly suited for this type of application, keeping past failures at gene therapy of these hemoglobinopathies in mind. Development, improvement, and methods for preparation of lentiviral-derived vectors are examined. Recently published results of successful gene therapy treatment of beta-thalassemic and sickle cell diseased mice using lentiviral-derived vectors are described. Finally, criticisms and future directions of lentiviral-based biotechnology are considered.     

From the item to the outcome: the promising prospects of PROMIS

Evaluation of patient reported outcomes, and in particular physical function, have gained increasing importance in research and therapy of patients with rheumatic diseases. Most instruments that are used for that purpose are rigid and suffer from floor and ceiling effects when used in patients whose physical function differs from the average. A new approach to the assessment of physical function uses computerised adaptive testing, by which precision and reliability of the measurement can be achieved for most patients, while even requiring less time for the assessment. Well calibrated and tested item and large item data banks are a prerequisite for this purpose, a process that is summarised in the present report by Bruce and colleagues.     

The role of the medial septum in the acoustic trachea of the cricket Gryllus bimaculatus

The auditory organs of the cricket which are situated in the front legs are joined together by a large transverse trachea which decisively influences their directional characteristics. The transverse trachea is medially divided by a septum. The importance of this septum for the localization of a sound source was tested by means of behavioural experiments in which the phonotactic movements of intact Gryllus bimaculatus females were compared quantitatively with those of the same specimen after perforation of the septum. The septal perforation does not noticeably influence locomotion in the absence of acoustic stimuli but selectively changes essential characteristics of phono taxis: 1) The animals walk in less straight lines. The oscillations around the mean course, typical of phonotaxis, are increased in amplitude, while the frequency decreases. 2) Course deviations from the direction of the sound source become more pronounced. 3) The threshold for phonotaxis is raised by about 10 dB. 4) Both the speed at which the animals walk and the proportion of time during which they are mobile are reduced. The results are discussed in relation to the role of the septum in the mechanism of sound localization, and with regard to its possible importance for the recognition of acoustic patterns.     

Echinococcus granulosus equinus: the histochemistry of the laminated layer of the hydatid cyst

The laminated layer of the hydatid cyst of E. granulosus equinus grown in mice has a polysaccharide substrate, which is not glycogen, but which possesses 1,2 glycols. This periodate-reactive component is demonstrated at the ultrastructural level using the thiosemicarbazide-silver proteinate technique which is also positive in Golgi-derived vesicles in the cytons and within the superficial cytoplasm at the parasite tissue/laminated layer interface. A carboxyl-acid mucopolysaccharide, lacking uronic and sialic acid residues, is present and weak reactions for sulphated acid mucopolysaccharide are recorded. The equivocal evidence for the presence of a chitin-like substrate is discussed. The predominantly basic protein component of the laminated layer has histochemically identifiable SS groups which are only very weakly demonstrated at the cytochemical level. Arginine and tryptophan are recorded. Lipid levels are low, and divalent cations, possibly calcium, are present.