Modulation by gibberellic acid and adenosine-3′,5′-cyclic monophosphate of starch hydrolysing activity of cowpea seedlings

In cowpea seedlings starch hydrolysing activity increases 35–50 fold on germination for 4 days. This increase in enzyme activity was inhibited by the in vivo addition of 1% glucose but this inhibition was completely overcome by the addition of gibberellic acid (GA₃) (10^?5 M) and adenosine-3′,5′-cyclic monophosphate (cAMP) (10^?5 M). At 5% glucose, GA₃ and cAMP were only partially effective. Structural analogues of cAMP failed to relieve the inhibitory effect of glucose. The inhibition by glucose is not direct but RNA and protein synthesis may be involved. Glucose appears to reduce the internal pool of cAMP which causes inhibition of RNA synthesis and decrease in starch hydrolysing activity. Exogenous application of cAMP may replenish the endogenous pool of cyclic nucleotide and thus overcome inhibition of RNA synthesis and enzyme activity.     

Calcium-induced proteolysis of spectrin and band 3 protein in rat erythrocyte membranes

Calcium-dependent protease activity capable of degrading a number of endogenous proteins was found in rat red blood cell membranes. This protease activity, like that found in human red blood cells, was activated by low concentrations of calcium, but in the rat red blood cells, unlike the human red blood cells, calcium-activated protease activity was membrane-bound. A number of endogenous membrane-bound proteins were degraded after the addition of calcium to the membranes. These included spectrin bands 1 and 2 as well as bands 3, 2.1, and 2.2. No calcium-induced aggregation (transglutaminase activity) was noted in the rat red blood cell membranes.     

Selective loss of myelin proteins during autolysis

Rat brains allowed to autolyze in situ for 12 h selectively lost myelin proteins. Basic proteins are markedly decreased, but DM-20 (1) and proteolipid proteins also are lost from the myelin of developing and mature rat brain. At room temperature there is a 50% decrease in the concentration of basic protein in myelin isolated from 15-day-old rats. Reducing the ambient temperature to 0°C reduces the loss to 20%. Similar but less marked changes occur in the brains of adult animals. The molar ratios of cholesterol, phospholipids, and glycolipids are unaffected by autolysis, and there is also no change in the specific activity of the myelin marker enzyme 2,3-cyclic nucleotide-3-phosphohydrolase (E.C.3.1.4.37). Electron microscopic examination of the isolated myelin demonstrates multilammelar structure with intraperiod lines.     

Apoptotic cell death of macrophages by iron-stressed <Emphasis Type="Italic">Salmonella enterica </Emphasis>serovar Typhimurium

Salmonella spp. have been shown to cause apoptosis of various host cell types as a part of their infection process. However, the induction of apoptosis remains to be looked into under the different host environments experienced by the pathogens. One of these is iron limitation, due to binding of iron in the host with proteins like lactoferrin, transferrin, haptoglobulin and hemoglobin etc. making non-availability of free iron to the pathogen for its growth and metabolism. In order to simulate the iron-limited in vivo situation, we studied the potential of Salmonella enterica serovar Typhimurium and its proteins under in vitro-created iron-stressed conditions, to cause apoptosis of macrophages (the first line of defence system). The apoptotic potential was evaluated qualitatively and quantitatively by various methods like assessment of nucleosomal DNA ladder (hallmark of apoptosis) and morphological evaluation by DNA intercalating dyes like acridine orange staining and Hoechst 33342-propidium iodide co-staining. It was observed that iron limitation could cause apoptotic cell death in a higher number of cells with the overexpression of proteins with subunit molecular weights of approximately 89, 54, 32 and 20 kDa. Salmonella may initiate apoptosis as a virulence strategy, but the death of host cells by the process of apoptosis rather than necrosis after getting a suicidal signal might be helpful for the host in order to save the surrounding cells, as well as to the parasite to enable it to spread systemically without inducing an inflammatory response.     

Minor alteration of microtubule dynamics causes loss of tension across kinetochore pairs and activates the spindle checkpoint

We have previously identified the opium alkaloid noscapine as a microtubule interacting agent that binds stoichiometrically to tubulin and alters its conformation. Here we show that, unlike many other microtubule inhibitors, noscapine does not significantly promote or inhibit microtubule polymerization. Instead, it alters the steady-state dynamics of microtubule assembly, primarily by increasing the amount of time that the microtubules spend in an attenuated (pause) state. Further studies reveal that even at high concentrations, noscapine does not alter the tubulin polymer/monomer ratio in HeLa cells. Cells treated with noscapine arrest at mitosis with nearly normal bipolar spindles. Strikingly, although most of the chromosomes in these cells are aligned at the metaphase plate, the rest remain near the spindle poles, both of which exhibit loss of tension across kinetochore pairs. Furthermore, levels of the spindle checkpoint proteins Mad2, Bub1, and BubR1 decrease by 138-, 3.7-, and 3.9-fold, respectively, at the kinetochore region upon chromosome alignment. Our results thus suggest that an exquisite control of microtubule dynamics is required for kinetochore tension generation and chromosome alignment during mitosis. Our data also support the idea that Mad2 and Bub1/BubR1 respond to kinetochore-microtubule attachment and/or tension to different degrees.     

Neurofilament protein synthesis and phosphorylation

Neurofilament proteins, a major intermediate filament component of the neuronal cytoskeleton, are organized as 10 nm thick filaments in axons and dendrites. They are large, abundantly phosphorylated proteins with numerous phosphate acceptor sites, up to 100 in some cases, organized as numerous repeat motifs. Together with other cytoskeletal components such as microtubules, MAPs, actin and plectin-like linking molecules, they make up a dynamic lattice that sustains neuronal function from neuronal “birthday” to apoptotic cell death. The activity of the neuronal cytoskeleton is regulated by phosphorylation, dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Factors regulating multisite phosphorylation of NFs are topographically localized, with maximum phosphorylation of NF proteins consigned to axons. Phosphorylation defines the nature of NF interactions with one another and with other cytoskeletal components such as microtubules, MAPs and actin. To understand how these functional interactions are regulated by phosphorylation we attempt to identify the relevant kinases and phosphatases, their specific targets and the factors modulating their activity. As an initial working model we propose that NF phosphorylation is regulated topographically in neurons by compartment-specific macromolecular complexes of substrates, kinases and phosphatases. This implies that axonal complexes differ structurally and functionally from those in cell bodies and dendrites. Such protein assemblies, by virtue of conformational changes within proteins, facilitate ordered, sequential multisite phosphorylations that modulate dynamic cytoskeletal interactions.     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, ^99mTc, and an aqueous marker, ¹²⁵I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped ¹²⁵I-labelled poly(vinyl pyrrolidone) were labelled with ^99mTc by the SnCl₂ method [2]. ^99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, ^99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than ¹²⁵I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60–70% of ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of ^99mTc-radioactivity was co-eluted with liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all ^99mTc-radioactivity does not represent the 60–70% of intact liposomes present in lymph nodes. Using the aqueous marker ¹²⁵I-labelled poly(vinyl pyrrolidone), the lymphnode localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that ¹²⁵I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier [2], where the authors used ^99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became ‘leaky’ to low-molecular-weight drugs, for example, methotrexate (M_r 454) to a much greater extent than with a large-molecular-weight substance, ¹²⁵I-labelled poly(vinyl pyrrolidone) (M_r 30 000–40 000), when incubated with rat lymph at 37°C. Using the two markers ^99mTc and ¹²⁵I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining λ-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with ^99mTc rather than using the SnCl₂ technique [2], or using other radionuclides as markers for γ-scan imaging.     

A Quantitative Analysis of Growth and Size Regulation in Manduca sexta: The Physiological Basis of Variation in Size and Age at Metamorphosis

Body size and development time are important life history traits because they are often highly correlated with fitness. Although the developmental mechanisms that control growth have been well studied, the mechanisms that control how a species-characteristic body size is achieved remain poorly understood. In insects adult body size is determined by the number of larval molts, the size increment at each molt, and the mechanism that determines during which instar larval growth will stop. Adult insects do not grow, so the size at which a larva stops growing determines adult body size. Here we develop a quantitative understanding of the kinetics of growth throughout larval life of Manduca sexta, under different conditions of nutrition and temperature, and for genetic strains with different adult body sizes. We show that the generally accepted view that the size increment at each molt is constant (Dyar’s Rule) is systematically violated: there is actually a progressive increase in the size increment from instar to instar that is independent of temperature. In addition, the mass-specific growth rate declines throughout the growth phase in a temperature-dependent manner. We show that growth within an instar follows a truncated Gompertz trajectory. The critical weight, which determines when in an instar a molt will occur, and the threshold size, which determines which instar is the last, are different in genetic strains with different adult body sizes. Under nutrient and temperature stress Manduca has a variable number of larval instars and we show that this is due to the fact that more molts at smaller increments are taken before threshold size is reached. We test whether the new insight into the kinetics of growth and size determination are sufficient to explain body size and development time through a mathematical model that incorporates our quantitative findings.