Experimental investigations in prolonged myocardial ischaemia. Note I. Biology of the myocardial fiber after transient myocardial fiber after transient myocardial ischaemia

The first ten days' evolution of post-ischaemic lesions of the premonitory or angina pectoris syndrome type was experimentally studied by the challenge of a short-term (10 and 15 min) ischaemia, of an adaptation to ischaemia and an adaptation followed by prolonged ischaemia (20 and 35 min). Worthy of note was the persistence of reversible lesions after short-term ischaemia and adaptation, and the progressive evolution towards cytolysis and cicatrization of some pancicellular foci after adaptation followed by prolonged ischaemia. The role of mitochondrial lesions, of lysosomal hydrolases, the inefficiency of renewed circulation, as well as problems of diagnosis are discussed.     

Q fever urban cases in Romania

Q fever urban sporadic cases in Romania in the 1981-87 period are reviewed as concerns their incidence, seasonality and epidemiological data. Urban cases represented 76.8% as against 23.2% rural cases from the 134 Q fever sporadic cases detected in this period. Cases were distributed throughout all months with peaks during April (18.4% of cases) and June (19.4%). Infections were not related to contact with livestock or domestic birds or with raw milk drinking. Cases could not be identified as Q fever occupational ones. 36.7% of patients were working in nonalimentary industry and only 12.6% were involved in meat industry, veterinary or human medical practices. New studies concerning unusual sources of urban infection such as dogs, cats or urban street pigeons are emphasized.     

Cell-membrane phospholipase C is involved in inducing the antiviral effect of interferon

A monospecific inhibitory antibody directed to phospholipase C (phosphoinositidase C) blocked the antiviral effect of human interferons alpha and beta when tested on human quiescent fibroblasts challenged with the vesicular stomatitis virus. This action was due to specific inhibition of polyphosphoinositide hydrolysis because (a) the F(ab)₂ fragment of the antibody molecule was also inhibitory; (b) excess antibodies directed to phospholipase A₂ and to a phosphatidylcholine-preferring phospholipase C did not have any inhibitory effect, and (c) the combination of 12-O-tetradecanoylphorbol-acetate and calcium ionophore A23187 had an interferon-like antiviral effect which was not influenced by the inhibitory anti-phospholipase C antibodies. To avoid an interferon-like effect due to induction of interferon by second messengers, Vero cells, which lack interferon biosynthesis, were also used. Liposomes containing inositol 1,4,5-triphosphate and 1-oleoyl-2-acetyl-rac-glycerol protected Vero cells against the infection with the vesicular stomatitis virus. These results taken together show that phosphoinositide-derived second messengers are involved in triggering the antiviral effect of interferons alpha and beta.     

Simulation of marked root hair curling in Rhizobium-legume symbiosis

The soil bacterium Rhizobium infects its leguminous host plants in temperate regions of the world mostly by way of the growing root hairs. Root hair curling is a prerequisite for root hair infection, although sidelong root hair infections occasionally have been observed. The processes underlying Rhizobium -induced root hair curling are unknown.
Computer simulation of root hair growth indicates that one-sided tip growth inhibition by Rhizobium can result in root hair curling when three conditions are simultaneously fulfilled: 1) rhizobial growth inhibition is strong enough to prevent removal out of the tip growth range: 2) root hair surface growth between the attached Rhizobium and the root hair top is inhibited; 3) rhizobial growth inhibition is limited to one side of the root hair.
The results predict that root hair curling by stimulation of tip growth is improbable. This study accentuates the need for information about the growth processes contributing to tip growth in leguminous root hairs.     

Dissimilation curves as a simple method for the characterization of growth of plant cell suspension cultures

A simple non-invasive method for the characterization of growth of a plant cell suspension in a single culture flask is given. The dissimilation of sugars by a cell-culture causes a loss of weight of the contents of the culture flask, and can therefore be used to follow the growth in that single culture flask. Because a correction for water evaporation is necessary, accurate results can only be obtained when a stable closure is used (e.g. Silicosen T-type plugs). The dissimilation curves obtained in this way were correlated to the concentration of sugars in the medium, the dry weight and the fresh weight. From these correlations the amount of intracellularly stored carbohydrates could be estimated. Rate constants for CO₂-diffusion were determined for different types of closure. These values allowed the estimation of CO₂ levels inside the culture flasks from the dissimilation curves (CO₂ release curves). The dissimilation curves obtained using this method can easily be related to other types of growth curves. Different growth-phases can be clearly distinguished, e.g. lag-phase, exponential growth-phase and stationary-phase.     

Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.     

Effects of protein kinase C inhibitors on viral entry and infectivity.

The protein kinase C inhibitor H-7 (2-20 microM) inhibited dose-dependently the infectivity of the vesicular stomatitis virus on cultured human fibroblasts. Electron microscopy showed that H-7 inhibited the viral entry. H-7 also inhibited the infectivity of four other enveloped viruses, herpes simplex I, turkey herpes, vaccinia and Sindbis. Similar results were obtained using staurosporine (2.5 nM), tamoxifen (40 microM), phloretin (140 microM), or W-7 (40 microM). However, the infectivity of non-enveloped viruses (e.g. poliomyelitis I) was not inhibited by H-7. These results show that protein kinase C is critically involved in the infectivity of enveloped viruses, most probably at the level of viral entry (receptor-mediated endocytosis).     

Cutting edge: C3, a key component of complement activation, is not required for the development of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis in mice

Experimental autoimmune encephalomyelitis (EAE), an inflammatory demyelinating disease of the CNS, is regarded as an experimental model for multiple sclerosis. The complement has been implicated in the pathogenesis of multiple sclerosis. To clarify the role of C in mouse EAE, we immunized mice deficient in C3 (C3(-/-)) and their wild-type (C3(+/+)) littermates with myelin oligodendrocyte glycoprotein peptide 35-55. C3(-/-) mice were susceptible to EAE as much as the C3(+/+) mice were. No differences were found for the production of IL-2, IL-4, IL-12, TNF-alpha, and IFN-gamma between C3(+/+) and C3(-/-) mice. This finding shows that C3, a key component in C activation, is not essential in myelin oligodendrocyte glycoprotein peptide-induced EAE in mice.     

Hypoxic rise in cytosolic calcium and renal proximal tubule injury mediated by a nickel-sensitive pathway

In the kidney, cell injury resulting from ischemia and hypoxia is thought to be due, in part, to increased cytosolic Ca(2+) levels, [Ca(2+)]i, leading to activation of lytic enzymes, cell dysfunction, and necrosis. We report evidence of a progressive and exponential increase in [Ca(2+)]i (from 245 +/- 10 to 975 +/- 100 nM at 45 mins), cell permeabilization and propidium iodide (PI) staining of the nucleus, and partial loss of cell transport functions such as Na(+)-gradient-dependent uptakes of (14)C-alpha-methylglucopyranoside and inorganic phosphate ((32)Pi) in proximal convoluted tubules of adult rabbits subjected to hypoxia. The rise in [Ca(2+)]i depended on the presence of extracellular [Ca(2+)] and could be blocked by 50 microM Ni(2+)but not by verapamil (100 microM). Presence of 50 microM Ni(2+) also reduced the hypoxia-induced morphological and functional injuries. We also used HEK 293 cells, a kidney cell line, incubated in media without glucose and exposed for 3.5 hrs to 1% O(2)-5% CO(2) and then returned to glucose-containing media for another 3.5 hrs in an air-5% CO(2) atmosphere and finally exposed for 1 min to media containing 1 microM PI. NiCl(2) (50 microM) or pentobarbital (300 microM) more than phenobarbital (1.5 mM), when present in the incubation medium during both the hypoxic and the reoxygenation periods, induced significant (P < 0.001) reductions in the number of cell nuclei stained with PI, similar to their relative potency as inhibitors of T channels. Our findings indicate that hypoxia-induced alterations in calcium level and subsequent cell injury in the proximal convoluted tubule and in HEK cells involve a nickel-sensitive and dihydropyridine insensitive pathway or channel.     

The erythropoietin receptor transmembrane domain mediates complex formation with viral anemic and polycythemic gp55 proteins

Erythropoietin receptor (EpoR) activation is crucial for mature red blood cell production. The murine EpoR can also be activated by the envelope protein of the polycythemic (P) spleen focus forming virus (SFFV), gp55-P. Due to differences in the TM sequence, gp55 of the anemic (A) strain SFFV, gp55-A, cannot efficiently activate the EpoR. Using antibody-mediated immunofluorescence co-patching, we show that the majority of EpoR forms hetero-oligomers at the cell surface with gp55-P and, surprisingly, with gp55-A. The EpoR TM domain is targeted by gp55-P and -A, as only chimeric receptors containing EpoR TM sequences oligomerized with gp55 proteins. Both gp55-P and gp55-A are homodimers on the cell surface, as shown by co-patching. However, when the homomeric interactions of the isolated TM domains were assayed by TOXCAT bacterial reporter system, only the TM sequence of gp55-P was dimerized. Thus, homo-oligomerization of gp55 proteins is insufficient for full EpoR activation, and a correct conformation of the dimer in the TM region is required. This is supported by the failure of gp55-A-->P, a mutant protein whose TM domain can homo-oligomerize, to fully activate EpoR. As unliganded EpoR forms TM-dependent but inactive homodimers, we propose that the EpoR can be activated to different extents by homodimeric gp55 proteins, depending on the conformation of the gp55 protein dimer in the TM region.