Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Effect of cultural conditions on the lipid profile of Thiobacillus ferroxidas

The intensitive investigations on the lipid profile of Thiobacillus ferrooxidans at various culture ages suggest some correlations of the lipid constitutents with the membrane-bound iron oxidation system. Phosphatidic acid, phosphatidyl serine and phosphatidyl ethanolamine were the major polar components; hydrocarbon, triglyceride and diglyceride were the main neutral components. Major fatty acids were C_16:0, C_16:1, C_16:3, C_18:1, C_18:3, C_22:1 while C_20:1, C_20:2, C_12:0, C_14:2, C_18:0, C_18:2, C_20:0, C_22:0 were found in trace amounts which also depended upon the phase of the growth. One lipoamino acid was identified as ornithine lipid in the polar fraction. Each and every component varied to some extent at different growth phasesindicating relationship of these lipids to the iron oxidation system of the strain.     

Manganese in cell metabolism of higher plants

Manganese, a group VII element of the periodic table, plays an important role in biological systems and exists in a variety of oxidation states. The normal level of Mn in air surrounding major industrial sites is 0.03 μg/m³, in drinking water 0.05 mg/liter and in soil between 560 and 850 ppm. Manganese is an essential trace element for higher plant systems. It is absorbed mainly as divalent Mn²⁺, which competes effectively with Mg²⁺ and strongly depresses its rate of uptake. The accumulation of Mn particularly takes place in peripheral cells of the leaf petiole, petiolule and palisade and spongy parenchyma cells. Mn is involved in photosynthesis and activation of different enzyme systems. Mn deficiency may be expressed as inhibition of cell elongation and yield decrease. Mn toxicity is one of the important growth limiting factors in acid soils. Plant tops are affected to a greater extent than root systems. The toxicity symptoms are, in general, similar to the deficiency symptoms. Toxic effects of Mn on plant growth have been attributed to several physiological and biochemical pathways, although the detailed mechanism is still not very clear. Higher O₂ uptake and loss of control in Mn activated enzyme systems have been associated with Mn toxicity. Mn interferes with the uptake, transport and use of several essential elements including Ca, Fe, Cu, Al, Si, Mg, K, P and N. Excess of Mn reduces the uptake of certain elements and increases that of others. pH plays an important role in Mn uptake. Acidic pH causes a lack of substantial amount of nitrate as an alternative electron acceptor and leads to a high amount of Mn in leaves. High microbial activity, water logging and poorly structured soils cause severe Mn toxicity even in neutral soils. The molecular mechanism of Mn-tolerance is not yet clear. The level of tolerance is different in different species and seems to be controlled by more than one gene. Further information is required on the factors affecting the distribution, accumulation and membrane permeability of the metal in different plant parts and different species. Understanding of the genetic basis of Mn-tolerance is necessary to improve adaptation of crops against acid soils, water logging and other adverse soil conditions.     

Are our actions aligned With our evidence? The skinny on changing the landscape of obesity

Recent debate about the role of food deserts in the United States (i.e., places that lack access to healthy foods) has prompted discussion on policies being enacted, including efforts that encourage the placement of full‐service supermarkets into food deserts. Other initiatives to address obesogenic neighborhood features include land use zoning and parks renovations. Yet, there is little evidence to demonstrate that such policies effect change. While we suspect most researchers and policymakers would agree that effective neighborhood change could be a powerful tool in combating obesity, we desperately need strong and sound evidence to guide decisions about where and how to invest.     

Site-specific conjugation of boron-containing dendrimers to anti-EGF receptor monoclonal antibody cetuximab (IMC-C225) and its evaluation as a potential delivery agent for neutron capture therapy

The gene encoding EGFR often is amplified in human gliomas, and the receptor itself has been considered as a potential target for the specific delivery of therapeutic agents to brain tumors. The purpose of the present study was to investigate the use of the chimeric MoAb cetuximab (IMC-C225), which is directed against EGFR and EGFRvIII, as a boron delivery agent for neutron capture therapy (NCT) of brain tumors. As determined by 125I-cetuximab radioligand binding assays, F98 rat glioma cells, which had been transfected with the gene encoding EGFR (F98EGFR), expressed 1.60 +/- 0.13 x 10(5) receptor sites/cell with a Ka = 1.64 +/- 0.32 x 10(8) M-1). F98 cells transfected with the gene encoding a mutant form of EGFR, designated the F98EGFRvIII glioma, expressed 1.07 +/- 0.10 x 10(5) receptor sites/cell with a Ka = 2.18 +/- 0.54 x 10(9) M-1 compared to background levels expressed on F98 wild-type cells (F98WT). A heavily boronated, fifth generation polyamidoamine (PAMAM or "starburst") dendrimer, G5-B1100, was linked to oligosaccharide moieties, which were distant from antigen binding sites of cetuximab, by means of the heterobifunctional reagents N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and N-(k-maleimidoundecanoic acid) hydrazide (KMUH). The resulting bioconjugate, designated C225-G5-B1100, was separated from the unconjugated dendrimer using a Sephacryl S-300 column. On the basis of the relative concentration ratios of boron and protein, there were approximately 1100 boron atoms per molecule of cetuximab with only a slight reduction of Ka. The localization of C225-G5-B1100 or G5-B1100 in rats bearing intracerebral implants of either F98EGFR or F98WT gliomas was determined 24 h following direct intratumoral (i.t.) injection at which time 92.3 +/- 23.3 micrograms B/g tumor was localized in F98EGFR gliomas versus 36.5 +/- 18.8 micrograms B/g tumor in F98WT gliomas and 13.4 +/- 6.1 micrograms in normal brain. In contrast, only 6.7 +/- 3.6 micrograms B/g tumor of G5-B1100 was localized in F98EGFR gliomas following i.t. injection, thereby demonstrating specific molecular targeting of EGFR. Based on these data, BNCT studies will be initiated in F98EGFR glioma bearing rats to evaluate C225-G5-B1100 for the treatment of intracerebral brain tumors.     

Stretch-induced Ca(2+) release via an IP(3)-insensitive Ca(2+) channel

Various mechanicalstimuli increase the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). A part of the increase in [Ca²⁺]_i isdue to the release of Ca²⁺ from intracellular stores. Wehave investigated the effect of mechanical stimulation produced bycyclical stretch on the release of Ca²⁺ from theintracellular stores. Permeabilized VSMC loaded with ⁴⁵Ca²⁺ were subjected to 7.5% average (15%maximal) cyclical stretch. This resulted in an increase in⁴⁵Ca²⁺ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP₃),ryanodine, and nicotinic acid adenine dinucleotide phosphate channels(NAADP) with 50 µg/ml heparin, 50 µM ruthenium red, and 25 µMthio-NADP, respectively, did not block the increase in⁴⁵Ca²⁺ efflux in response to cyclical stretch.However, 10 µM lanthanum, 10 µM gadolinium, and 10 µMcytochalasin D but not 10 µM nocodazole inhibited the increase in⁴⁵Ca²⁺ efflux. This supports the existence of anovel stretch-sensitive intracellular Ca²⁺ store in VSMCthat is distinct from the IP₃-, ryanodine-, and NAADP-sensitive stores.

     

What does it take to be a plant pathogen: genomic insights from <Emphasis Type="Italic">Streptomyces</Emphasis> species

Plant pathogenicity is rare in the genus Streptomyces, with only a dozen or so species possessing this trait out of the more than 900 species described. Nevertheless, such species have had a significant impact on agricultural economies throughout the world due to their ability to cause important crop diseases such as potato common scab, which is characterized by lesions that form on the potato tuber surface. All pathogenic species that cause common scab produce a family of phytotoxins called the thaxtomins, which function as cellulose synthesis inhibitors. In addition, the nec1 and tomA genes are conserved in several pathogenic streptomycetes, the former of which is predicted to function in the suppression of plant defense responses. Streptomyces scabies is the oldest plant pathogen described and has a world-wide distribution, whereas species such as S. turgidiscabies and S. acidiscabies are believed to be newly emergent pathogens found in more limited geographical locations. The genome sequence of S. scabies 87-22 was recently completed, and comparative genomic analyses with other sequenced microbial pathogens have revealed the presence of additional genes that may play a role in plant pathogenicity, an idea that is supported by functional analysis of one such putative virulence locus. In addition, the availability of multiple genome sequences for both pathogenic and nonpathogenic streptomycetes has provided an opportunity for comparative genomic analyses to identify the Streptomyces pathogenome. Such genomic analyses will contribute to the fundamental understanding of the mechanisms and evolution of plant pathogenicity and plant-microbe biology within this genus.     

Direct and callus-mediated protocorm-like body induction from shoot-tips of <Emphasis Type="Italic">Dendrobium chrysotoxum</Emphasis> Lindl. (Orchidaceae)

An efficient method of mass propagation of Dendrobium chrysotoxum Lindl. was developed using a shoot-tip culture system. Both direct and callus-mediated formation of protocorm-like bodies (PLBs) occurred from the basal cut surface of explants. Frequency of callusing was best in the presence of 2 μM thidiazuron (TDZ) or N⁶-benzylaminopurine (BAP). The callus exhibited complete hormone autonomy for growth and differentiation of PLBs and was maintained for 18 months without any exogenous growth regulators, an aspect important for minimising somaclonal variation. However, the rate of callus growth and PLB formation varied with application of cytokinin and auxin. In addition, the callus exhibited a differential sensitivity to the exogenous cytokinins. While BAP promoted callus growth and PLB differentiation, TDZ was inhibitory to callus mediated PLB formation and caused extensive necrosis of callus. Although α-naphthaleneacetic acid (NAA) had no significant effect on the induction of callus, subsequent growth was best in its presence. Using a 3-month subculture period, a 69-fold increase in callus weight was achieved with 0.5 μM NAA, while as many as 133 PLBs could be obtained per 100 mg callus in the presence of 1 μM NAA. For direct PLB formation, the optimum cytokinin dosage was dependent upon the type of cytokinin used. While TDZ was most effective at a concentration of 1 μM (15 PLBs per explant), for similar PLB yield the application of 8 μM BAP was essential.     

Proteolysis of insulin-like growth factor binding proteins (IGFBPs) by calpain

Calpains are non-lysosomal, Ca 2+ -dependent cysteine proteases, which are ubiquitously distributed across cell types and vertebrate species. The rules that govern calpain specificity have not yet been determined. To elucidate the cleavage pattern of calpains, we carried out calpain-induced proteolytic studies on the insulin-like growth factor binding proteins IGFBP-4 and -5. Proteolysis of IGFBPs is well characterized in numerous reports. Our results show that calpain cleavage sites are in the non-conserved unstructured regions of the IGFBPs. Compilation of the calpain-induced proteolytic cleavage sites in several proteins reported in the literature, together with our present study, has not revealed clear preferences for amino acid sequences. We therefore conclude that calpains seem not to recognize amino acid sequences, but instead cleave with low sequence specificity at unstructured or solvent-exposed fragments that connect folded, stable domains of target proteins.