Insulin depletion leads to adipose-specific cell death in obese but not lean mice

Mutation of the obese gene produces obesity, hyperinsulinemia, and compensatory "overexpression" of the defective gene. As insulin activates obese gene expression, it seemed possible that hyperinsulinemia might be responsible for overexpression of the gene. To address this question we rapidly neutralized circulating insulin by injection of an insulin antibody. Unexpectedly, insulin depletion in obese (ob/ob or db/db) mice caused massive adipose RNA degradation confirmed by histological analysis to result from adipocyte cell death by a largely necrotic mechanism. This effect was not observed in lean littermates and was completely corrected by coadministration of insulin. Comparison of multiple tissues demonstrated that the effect was restricted to adipose tissue. Insulin depletion in obese mice by administration of streptozotocin also led to cell death, but this death was less extensive and appeared to be apoptotic in mechanism. Thus insulin may promote the survival side of the physiological balance between adipocyte survival and death.     

Binding and lysing of blood clots using MRX-408

RATIONALE AND OBJECTIVES: A thrombus-specific ultrasound contrast agent, MRX-408, has been developed recently. This agent consists of phospholipid-coated microbubbles with a ligand capable of targeting the GPIIb/IIIa receptor, thereby allowing the microbubbles to bind with thrombi rich in activated platelets. In vitro and in vivo animal experiments have been conducted to examine imaging enhancement and sonothrombolysis using this agent compared with a nontargeted agent. METHODS: For clot binding, blood-smeared slides were incubated with microbubbles and examined under a light microscope. Change in backscatter signals from the blood clots after binding was examined by both an ultrasound scanner and two single-element transducers arranged in a transmitter-receiver pair. For clot lysis, either 1-MHz or 20-KHz ultrasound was used to enhance the lysing effects of MRX-408 with or without urokinase. RESULTS: Evidence of binding was demonstrated under a microscope. In vitro experiments showed that the "acoustic signature", or properties, of blood clots changed after binding. Clots became more echogenic and nonlinear. In vivo fundamental ultrasound imaging confirmed that as a result of binding, blood clots were more visible, the area of detection was improved, and shadowing behind clots was more noticeable. Under 1-MHz ultrasound and 30 minutes of treatment, lysis efficiency reached 34% with MRX-408, whereas there was no visible clot lysis with saline. CONCLUSION: The results of these preliminary studies show that as a contrast agent, MRX-408 enhanced clots under ultrasound imaging and facilitated sonothrombolysis with or without thrombolytic drugs.     

Auditory system plasticity in children after long periods of complete deafness

Separation and identification of proteins by two-dimensional (2-D) electrophoresis can be used for protein-based gene expression analysis. In this report single protein spots, from polyvinylidene difluoride blots of micropreparative E. coli 2-D gels, were rapidly and economically identified by matching their amino acid composition, estimated pI and molecular weight against all E. coli entries in the SWISS-PROT database. Thirty proteins from an E. coli 2-D map were analyzed and identities assigned. Three of the proteins were unknown. By protein sequencing analysis, 20 of the 27 proteins were correctly identified. Importantly, correct identifications showed unambiguous "correct" score patterns. While incorrect protein identifications also showed distinctive score patterns, indicating that protein must be identified by other means. These techniques allow large-scale screening of the protein complement of simple organisms, or tissues in normal and disease states. The computer program described here is accessible via the World Wide Web at URL address (http:@expasy.hcuge.ch/).     

Laparoscopic versus open appendectomy

BACKGROUND: Clinical manifestations and course of sickle-cell anemia are variable. Knowledge about the factors, possibly geographic, that influence prognosis are still scanty. POPULATION AND METHODS: Data of hospitalization and management of children with sickle-cell disease were studied during two years (1992-1993) in the Pediatric Unit of Libreville Hospital. They concerned 205 admissions of 171 children and 131 outpatients. RESULTS: The main causes of hospitalization were: acute anemia (36 cases before the age of 5 years); painful crisis whose frequency increased with age (23% before 5 years, 35% between 5 and 10, 42% after 10 years); infections, essentially pulmonary occurring early, and bone infections at any age. Eight children died (because a complication of their disease). Among the 131 outpatients, half were detected because pyrexia, anemia and/or more often "hand-foot syndrome". More than 60% had hepatomegaly, one third still had splenomegaly after five years of age and more than one third was icteric. More than half children older than ten years had growth disorders. Mean hemoglobin level was 7 g/dL. 21 of the 83 tested children for HBsAg were positive and only one out of 79 was positive for HIV. CONCLUSIONS: Clinical manifestations and course of sickle-cell anemia in our patients are similar to those reported in Congolese children. Genetic and environmental factors may be responsible for differences with children from other, in particular French, cohorts.     

Metabolic role of glutamine and its importance in nutritional therapy

The advances in the field of nutritional support have made certain nutrients very relevant, which, although they have been known for a long time, at present represent an important chapter in nutrition, entering into what is known as "nutritional pharmacology". Among these nutrients is glutamine, an amino acid classified as non-essential, but which in certain circumstances may become to be considered as an "essential nutrient". In the present review, a review is made of its metabolic role, synthesis and degradation, metabolic routes and functions under normal conditions as well as under critical conditions. It is known that glutamine stimulates the synthesis and inhibits the degradation of proteins, it is an important vehicle for the transport of nitrogen and carbon within the tissues, it stimulates the synthesis of hepatic glycogen, it is an energy source for cell division, for the growth of different cells of rapid replication, such as enterocytes, colonocytes, and fibroblasts, as well as for other cells of the immune system, such as lymphocytes and macrophages. Thus its role in the maintenance of structure, in metabolism and function of the intestinal mucosa, and in dysfunctions of the immune system. In parenteral nutrition, at present there are no preparations which include it, given the stability problems which it presents, although attempts have been made to resolve this, using different possibilities, such as di-peptides. However, in enteral nutrition, the diets tend to include it, although in a small proportion. Nevertheless, having recognized its beneficial role in a certain type of patients, at present there are diets which contain glutamine in higher doses, with the object of attempting to cover the increased demands of glutamine which shall arise in these situations. The inclusion of glutamine in nutritional therapy is supported by multiple studies which reflect the beneficial effect of this nutrient, both in enteral nutrition as in parenteral nutrition.     

Supramalleolar osteotomy for ankle valgus in myelomeningocele

Oral fibroblasts stimulated invasion of oral-carcinoma cells into the collagen matrix. The mechanisms of the fibroblast-induced stimulation of invasiveness was further investigated by examining cell motility and proteolytic activity of tumor cells, using mainly an adenoid-cystic-carcinoma cell line (ACCS) and normal fibroblasts from gingival tissues. Conditioned medium from the fibroblasts grown in serum-free medium was fractionated on a Superdex 200 pg column, and Peak 1 eluted at 200 to 300 kDa and Peak 2 eluted at 50 to 100 kDa were found to contain different specific activity. Treatment of ACCS cells with Peak 1 resulted in an increase in the production of proteolytic enzymes. Peak 2 stimulated both chemotaxis and chemokinesis of ACCS cells. A chemotactic factor was purified from the heparin-unbound fraction of Peak 2 by anion exchange and hydrophobic chromatography, and was named "fibroblast-derived motility factor (FDMF)". At 1 microg/ml, FDMF stimulated chemotaxis of ACCS cells by 4-fold compared with unstimulated controls. Characterization of the physicochemical properties of FDMF suggested that it might be different from any known motility factors. Exposure of ACCS cells to FDMF resulted in reduced amounts of actin stress fiber in the cytoplasm and induction of tyrosine phosphorylation of several cellular proteins detectable 30 to 60 min after treatment. These FDMF-induced changes were blocked by pre-treatment either with genistein or with pertussis toxin. These findings suggest that FDMF may be a novel protein which stimulates cell motility via a signaling pathway mediated by a pertussis-toxin-sensitive G protein and tyrosine phosphorylation.     

Structure and evolutionary implications of a "cysteine-rich" Campoletis sonorensis polydnavirus gene family

For successful parasitization, the female Campoletis sonorensis endoparasitic wasp injects a polydnavirus into its host, Heliothis virescens, during oviposition. Viral gene expression induces immunosuppression and alters development of the host. We report here that three abundantly expressed genes, VHv1.1, WHv1.0, and WHv1.6, describes a polydnavirus "cysteine-rich" gene family which may be important in inducing these host manifestations. These genes have a similar primary gene structure and their proteins contain cysteine motifs characteristic of snail ion-channel ligands, the omega-conotoxins. Like the omega-conotoxins, the intercysteine amino acid residues are hypervariable with only three identical amino acids in all motifs. The conservation of this domain in the three viral genes may reflect an important functional role for these viral proteins in the parasitization of H. virescens. The three genes also contain introns similar in sequence at comparable positions in their 5' untranslated leaders and coding sequences. VHv1.1 contains two cysteine motifs, and each motif is interrupted by an intron at the same position as in the cysteine motifs of WHv1.0 and WHv1.6. Intron 2 sequences of WHv1.0 and WHv1.6 are 92% identical, while the immediately flanking exon sequences encoding the cysteine motifs are only 76% identical. This provides an example of nuclear pre-mRNA introns which are more conserved than flanking exons among members of a gene family.     

Fasciola hepatica development in the experimentally infected black rat Rattus rattus

Coat colour changes in polar animals are related to seasonal variation in photic inputs. The present work was performed to study the photoresponses of hair follicular melanocytes in human skin. The melanocytes, being photosensitive cells, can function as UV biosensors, since dendrites extend towards the source of UV light. Fifty-one skin biopsies from the margin of vitiligo were subjected to whole skin organ cultures. These were exposed to a pulse of UV light to study hair bulb melanocytes in vitiligo. It is observed that the melanocytes are seen within the anagen matrix. These melanocytes are poorly dendritic in control and dark-incubated cultures. On UV exposure, they become highly dendritic, the dendrites extending towards the hair shaft in 93.5%. They show prominent catechol oxidase and noradrenaline positivity, all features of UV responsiveness. The melanocytes within the hair follicle are not directly exposed to UV light. The melanocyte dendricity and the alignment of dendrites towards the shaft on UV exposure indicate that the columns of the cells in the hair shaft act as an efficient fibre-optic system, transmitting UV light. Morphologically, the keratinocytes in the hair shaft are arranged in compressed linear columns which resemble the coaxial bundles of commercial fibre-optic strands as is observed in plants. Keratinocytes in the inner and outer sheaths do not show this arrangement. Thus the hair follicle functions as a specialised UV receptor in the skin responding to nuances of photic inputs in human skin. This is reflected in coat colour changes in animals exposed to large variations in day-night cycles.     

A Novel Mouse Model of TGFβ2-Induced Ocular Hypertension Using Lentiviral Gene Delivery

Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) β2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFβ2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFβ2 in the TM. We developed an LV vector-encoding active hTGFβ2^C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFβ2^C226,228S. We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFβ2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFβ2^C226,228S exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFβ2^C226,228S that induces TM dysfunction and outflow resistance.