Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.     

Endoscopic ultrasonography in the local staging of primary gastric lymphoma

Endoscopic ultrasonography (EUS) and endoscopy were prospectively performed and compared to the histopathologic findings of the resection specimens in 24 patients with primary gastric lymphoma (PGL). On EUS, three types of PGL could be differentiated, a superficial type (n = 10), an infiltrating type (n = 12) and a tumorous type (n = 2). In the correct assessment of surface extension of the tumors, endoscopy and EUS agreed in 37.5% of cases and EUS showed more extensive disease than endoscopy in 58% of cases. However, in comparison to the resection specimens, EUS still underestimated the tumor surface extension in 37.5% of cases; this was mainly in low grade malignant PGL. The depth of tumor infiltration was correctly determined on EUS compared to the resection specimens in 91.5% of cases. Sensitivity, specificity and accuracy of diagnosing lymph node metastases were 100%, 80% and 83%, respectively. We conclude that EUS is a useful pre-therapeutic staging tool for primary gastric lymphoma but there remain some problems in determining the longitudinal and circular tumor spread in order to accurately guide the extent of gastric resection.     

Theoretical explanation of the relationship between backscattered electron and x-ray linear attenuation coefficients in calcified tissues

Adverse reactions to seafood are common and may cause many types of symptoms that are difficult to define. The nature of these reactions are variable including allergic and toxic reactions as well as infectious diseases. The differentiation between these entities is essential in choosing therapy. We describe 9 patients with IgE mediated allergic reactions due to crustaceans and fish diagnosed from case history, clinical findings, skin tests and specific IgE antibodies. Most symptoms of a IgE mediated allergic reaction appear within 30 minutes after ingestion. Characteristics clinical features may include an urticarial rash, gastrointestinal symptoms and even anaphylaxis. In this case immediate therapy with intravenous glucocorticoids, antihistamine and perhaps subcutaneous epinephrine is required.     

Interpersonal and self-reported hostility among combat veterans with and without posttraumatic stress disorder

Despite the low concentrations of heavy metals in the surrounding medium, aquatic organisms take them up and accumulate them in their soft tissues to concentrations several fold higher than those of ambient levels (Bryan 1979; Rainbow et al. 1990). Knowledge of accumulation patterns of a particular trace metal is a prerequisite for understanding the significance of an observed metal concentration in a particular animal, especially from the aspect of biomonitoring. Many marine invertebrates accumulate heavy metals without any regulation and the accumulation necessarily being associated with mechanisms to store the metals in a detoxified form. Two detoxification mechanisms have been described, both of which may occur in one specimen. Heavy metals can either be bound up in insoluble metalliferous 'granules' (Mason and Nott 1981), or are bound to soluble metal-binding ligands, such as metallothioneins (Roesijadi 1992). Some marine decapod crustaceans have an innate ability to regulate the internal concentrations of essential but potentially toxic metals within a constant level, presumably to meet their metabolic demands (Rainbow 1985, 1992). However, at present, there is no such information relating to freshwater decapod crustaceans, especially shrimps which occupy a totally different environment. Macrobrachium malcolmsonii (Milne Edwards), a potential aquaculture species for freshwater is found in abundance in one of the major Indian rivers, the Cauvery. In the present study, an attempt was made to determine whether the freshwater prawn, M. malcolmsonii, is able to regulate the three essential elements, copper, chromium and zinc, over a wide range of dissolved concentrations. These three metals were chosen because the Cauvery River receives pollutants containing these metals (Vijayram et al. 1990).     

Sulphonylureas do not increase insulin secretion by a mechanism other than a rise in cytoplasmic Ca2+ in pancreatic B-cells

The following sequence of events is thought to underlie the stimulation of insulin release by hypoglycaemic sulphonylureas. Interaction of the drugs with a high-affinity binding site (sulphonylurea receptor) in the B-cell membrane leads to closure of ATP-sensitive K+ channels, depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx and rise in cytoplasmic [Ca2+]i. Recent experiments using permeabilized islet cells or measuring changes in B-cell membrane capacitance have suggested that sulphonylureas can increase insulin release by a mechanism independent of a change in [Ca2+]i. This provocative hypothesis was tested here with intact mouse islets. When B-cells were strongly depolarized by 60 mM K+, [Ca2+]i was increased and insulin secretion stimulated. Under these conditions, tolbutamide did not further increase [Ca2+]i or insulin release, whether it was applied before or after high K+, and whether the concentration of glucose was 3 or 15 mM. This contrasts with the ability of forskolin and phorbol 12-myristate 13-acetate (PMA) to increase release in the presence of high K+. Tolbutamide also failed to increase insulin release from islets depolarized with barium (substituted for extracellular Ca2+) or with arginine in the presence of high glucose. Glibenclamide and its non-sulphonylurea moiety meglitinide were also without effect on insulin release from already depolarized B-cells. In the absence of extracellular Ca2+, acetylcholine induced monophasic peaks of [Ca2+]i and insulin secretion which were both unaffected by tolbutamide. Insulin release from permeabilized islet cells was stimulated by raising free Ca2+ (between 0.1 and 23 microM). This effect was not affected by tolbutamide and inconsistently increased by glibenclamide. In conclusion, the present study does not support the proposal that hypoglycaemic sulphonylureas can increase insulin release even when they do not also raise [Ca2+]i in B-cells.