Nuclear transfer of perchloric acid-soluble protein by endoplasmic reticulum stressors

Perchloric acid-soluble protein (PSP) is highly conserved during evolution from bacteria to mammals. Although PSP has been recognized as an inhibitor of translation and proliferation in vitro, its precise biological role has not yet been elucidated. Since we previously found similar distributions for PSP and the endoplasmic reticulum (ER) and Golgi complex, the intracellular distribution of PSP was analyzed in more detail. Immunofluorescence studies indicated that PSP co-localized with the ER and Golgi complex, since the distribution pattern of PSP was well matched to both of these organelles. An immunoelectron microscopic study revealed PSP was located not only in the cytosol but also on the surface of the outer ER membrane. Since PSP was present on the ER, we speculated that it may be associated with ER function. Therefore, we analyzed whether or not the ER stress response, which is one of the ER functions, affected PSP expression. The results showed that various ER stressors (thapsigargin, A23187, tunicamycin, brefeldin A, and cisplatin) provoked a dramatic change in the localization of PSP from outside of the nucleus to inside the nucleus within 3 h. Moreover, the ER stressors induced PSP expression. These results suggest that PSP is involved in the cellular response to ER stressors, and that the change in localization of PSP from the ER to the nucleus may be associated with ER stress responses.     

Mapping of the binding sites involved in PSP94–CRISP-3 interaction by molecular dissection of the complex

Background

Human Prostate Secretory Protein of 94 amino acids (PSP94) has been shown to bind human CRISP-3 (cysteine-rich secretory protein 3) with very high affinity. CRISP-3 belongs to the CRISP family of proteins having a PR-1 (pathogenesis related protein 1) domain at its N-terminal and ion channel regulatory (ICR) domain at its C-terminal connected by a hinge region. Functional significance of this complex is not yet known.

Methods

In order to identify the residues and/or regions involved in PSP94–CRISP-3 interaction, site-directed mutagenesis was employed. Effect of the mutations on the interaction was studied by co-immunoprecipitation (Co-IP).

Results

For PSP94, amino acids Y³, F⁴, P⁵⁶ and the C-terminal β-strand were found to be crucial for interacting with CRISP-3. A disulfide bond between the two domains of PSP94 (C³⁷A–C⁷³A) was also important for this interaction. In case of CRISP-3, the N-terminal domain alone could not maintain a strong interaction with PSP94 but it required presence of the hinge region and not the C-terminal domain. Apart from CRISP-3, CRISP-2 was also found to interact with human PSP94. Based on our findings the most likely model of PSP94–CRISP-3 complex has been proposed.

Conclusion

The terminal β-strands of PSP94 contact the first α-helix and the hinge region of CRISP-3.

General significance

Involvement of the hinge region of CRISPs in interaction with PSP94 may affect the domain movement of CRISPs essential for the ion-channel regulatory activity resulting in inhibition of this activity.     

T-cell responses at the host: tumour interface

The evidence considered here reinforces the conclusion that T-cell responses to tumours involve complex cellular interactions. An attempt to summarize some of these interactions is shown. This emphasizes that not only are the interactions between the effector cell populations complicated, but that the target cell surface is also subject to variation and modification as a result of the immune response. A feature that also emerges from these studies is that most cells apparently responding to or infiltrating a tumour do not necessarily participate in its destruction, and it is in this area that experimental tumour systems have particular value. This also perhaps explains the preoccupation of experimentalists with the identification of 'the' effector cell crucial to tumour rejection. However, there is heterogeneity between systems in terms of the type of rejection response induced, but a logical basis for this heterogeneity is not established. If experimental studies could define the nature of the immune response generated by a tumour in the context of the biological features of the tumour itself, this could lead to the prediction of the immunogenicity and potential for induction of a rejection response for that tumor. Clearly, experimental tumour systems do not provide an exact reflection of the situation with human tumours. However, they may provide systems that illuminate particular aspects of the human response, and give precedents to guide the interpretation of data derived from human systems. This form of assessment is still at an early stage, but developments in the experimental field should provide a framework for the development and exploitation of T-cell responses to tumours.     

k-Carrageenan Gel as Agent to Sequester Paralytic Shellfish Poison

The action of k-carrageenan gel to sequester paralytic shellfish poison (PSP) was tested and characterized. When an extract from a Philippine strain of Pyrodinium bahamense var. compressum was used as PSP solution, the PSP-sequestering property of k-carrageenan gel was found to be dependent on gel surface area, interaction time, and polysaccharide concentration. The interaction was also found to be affected by high concentrations of monovalent cations. The characteristics of k-carrageenan as a PSP-sequestering agent all point to cation exchange as its mechanism of action. It is also proposed that the polysaccharide gel can be utilized as an agent to alleviate PSP intoxication.     

Role of desensitized acetylcholine receptors in the development of post-tetanic potentiation

A hypothesis that desensitized acetylcholine (ACh) receptors are responsible for postsynaptic potentiation (PSP) was verified in experiments performed on the sartorial muscle of the frog using a voltage-clamp recording technique. The PSP was estimated, at active and inhibited acetylcholinesterase, with the use of paired stimulation of the motor nerve at various interpulse intervals, according to an increase in the time constant () of the second end-plate current (EPC) relative to that of the first EPC. The appearance of desensitized receptors on the postsynaptic membrane, caused by the application of a promoter of desensitization proadiphen or by application of exogenous ACh, was followed by acceleration of the decay of the first EPC and by a decrease in its amplitude. However, this did not intensify PSP, which even decreased in the presence of proadiphen under the influence of ACh and membrane hyperpolarization. A sharp increase in calcium concentration after certain period of ACh influence, which increased quantum ACh release, did not enhance PSP, which allows us to suggest the role of the presynaptic factor to be insignificant. The results provide arguments against the direct involvement of desensitized acetylcholine receptors in the PSP development.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 361–367, September–December, 1995.     

A theoretical and empirical investigation of delayed growth response in the continuous culture of bacteria

When the growth of bacteria in a chemostat is controlled by limiting the supply of a single essential nutrient, the growth rate is affected both by the concentration of this nutrient in the culture medium and by the amount of time that it takes for the chemical and physiological processes that result in the production of new biomass. Thus, although the uptake of nutrient by cells is an essentially instantaneous process, the addition of new biomass is delayed by the amount of time that it takes to metabolize the nutrient. Mathematical models that incorporate this "delayed growth response" (DGR) phenomenon have been developed and analysed. However, because they are formulated in terms of parameters that are difficult to measure directly, these models are of limited value to experimentalists. In this paper, we introduce a DGR model that is formulated in terms of measurable parameters. In addition, we provide for this model a complete set of criteria for determining persistence versus extinction of the bacterial culture in the chemostat. Specifically, we show that DGR plays a role in determining persistence versus extinction only under certain ranges of chemostat operating parameters. It is also shown, however, that DGR plays a role in determining the steady-state nutrient and bacteria concentrations in all instances of persistence. The steady state and transient behavior of solutions of our model is found to be in agreement with data that we obtained in growing Escherichia coli 23716 in a chemostat with glucose as a limiting nutrient. One of the theoretical predictions of our model that does not occur in other DGR models is that under certain conditions a large delay in growth response might actually have a positive effect on the bacteria's ability to persist.     

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.     

Plasmatocyte spreading peptide (PSP1) and growth blocking peptide (GBP) are multifunctional homologs

Recently, we identified Plasmatocyte spreading peptide (PSP1) from the moth Pseudoplusia includens and reported that it mediates adhesion of hemocytes to foreign surfaces. PSP1 is structurally very similar to three classes of peptides identified earlier from other species of Lepidoptera: growth blocking peptide (GBP) originally identified in Pseudaletia separata, and a series of related peptides from other species designated as paralytic (PP) or cardioactive (CAP) peptides. In this study, we conducted parallel experiments in P. includens and P. separata to determine whether PSP1 and GBP have distinct or multiple biological activities. Both peptides affected the adhesive state of hemocytes from each moth very similarly. PSP1 and GBP exhibited significant growth blocking and paralytic activity in P. separata. Both peptides also had growth blocking activity in P. includens although larvae had to be injected with higher doses of each peptide to reduce weight gain than was observed for P. separata. However, GBP and PSP1 had little paralytic activity in P. includens. Collectively, our results indicate that GBP and PSP1 are multifunctional, but that some interspecific variation also exists in their growth blocking and paralytic activities. We suggest that all PSP1, GBP, PP and CAP family members are homologs that likely have multiple biological activities. Based upon the unique consensus sequence of their N termini, we propose that these molecules be henceforth referred to as members of the "ENF" peptide family.     

Alanine-scanning mutagenesis of plasmatocyte spreading peptide identifies critical residues for biological activity

Plasmatocyte spreading peptide (PSP) is a 23-amino acid cytokine that induces a class of insect immune cells called plasmatocytes to spread on foreign surfaces. The structure of PSP consists of a disordered N terminus (residues 1-6) and a well-defined core (residues 7-23) stabilized by a disulfide bridge between Cys(7) and Cys(19), hydrophobic interactions, and a short beta-hairpin. Structural comparisons also indicate that the core region of PSP adopts an epidermal growth factor (EGF)-like fold very similar to the C-terminal subdomain of EGF-like module 5 of thrombomodulin. To identify residues important for plasmatocyte spreading activity, we bioassayed PSP mutants in which amino acids were either replaced with alanine or deleted. Within the well-defined core of PSP, alanine replacement of Cys(7) and Cys(19) (C7.19A) eliminated all activity. Alanine replacement of Arg(13) reduced activity approximately 1000-fold in comparison to wild-type PSP, whereas replacement of the other charged residues (Asp(16), Arg(18), Lys(20)) surrounding Cys(19) diminished activity to a lesser degree. The point mutants Y11A, T14A, T22A, and F23A had activity identical or only slightly reduced to that of wild-type PSP. The mutant PSP-(7-23) lacked the entire unstructured domain of PSP and was found to have no plasmatocyte spreading activity. Surprisingly, E1A and N2A had higher activity than wild-type PSP, but F3A had almost no activity. We thus concluded that the lack of activity for PSP-(7-23) was largely due to the critical importance of Phe(3). To determine whether reductions in activity correlated with alterations in tertiary structure, we compared the C7.19A, R13A, R18A, and F3A mutants to wild-type PSP by NMR spectroscopy. As expected, the simultaneous replacement of Cys(7) and Cys(19) profoundly affected tertiary structure, but the R13A, R18A, and F3A mutants did not differ from wild-type PSP. Collectively, these results indicate that residues in both the unstructured and structured domains of PSP are required for plasmatocyte-spreading activity.     

Quantum-like brain: "Interference of minds"

We present a contextualist statistical realistic model for quantum-like representations in physics, cognitive science, and psychology. We apply this model to describe cognitive experiments to check quantum-like structures of mental processes. The crucial role is played by interference of probabilities for mental observables. Recently one such experiment based on recognition of images was performed. This experiment confirmed our prediction on the quantum-like behavior of mind. In our approach "quantumness of mind" has no direct relation to the fact that the brain (as any physical body) is composed of quantum particles. We invented a new terminology "quantum-like (QL) mind." Cognitive QL-behavior is characterized by a nonzero coefficient of interference lambda. This coefficient can be found on the basis of statistical data. There are predicted not only cos theta-interference of probabilities, but also hyperbolic cosh theta-interference. This interference was never observed for physical systems, but we could not exclude this possibility for cognitive systems. We propose a model of brain functioning as a QL-computer (there is a discussion on the difference between quantum and QL computers).     

Mechanistic modeling confronts the complexity of molecular cell biology

Mechanistic modeling has the potential to transform how cell biologists contend with the inescapable complexity of modern biology. I am a physiologist–electrical engineer–systems biologist who has been working at the level of cell biology for the past 24 years. This perspective aims 1) to convey why we build models, 2) to enumerate the major approaches to modeling and their philosophical differences, 3) to address some recurrent concerns raised by experimentalists, and then 4) to imagine a future in which teams of experimentalists and modelers build—and subject to exhaustive experimental tests—models covering the entire spectrum from molecular cell biology to human pathophysiology. There is, in my view, no technical obstacle to this future, but it will require some plasticity in the biological research mind-set.     

Differential expression of PSP94 in rat prostate lobes as demonstrated by an antibody against recombinant GST-PSP94.

Prostate secretory protein (PSP94, 94 amino acids) is one of the most abundant proteins secreted from the prostate. Its biological role is unknown and still controversial, although it is assumed to have the potential to be a biomarker and a suppressor of prostate cancer. In order to establish an animal model to further elucidate its biological role, we expressed the mature form of rat PSP94 in Escherichia coli, using a glutathione S-transferase (GST) fusion expression vector; we generated a polyclonal rabbit antibody against the recombinant protein. The antibody specifically recognized recombinant rat PSP94 and cross-reacted only very weakly with its human homologue. Using the characterized anti-rat PSP94 antibody, we found that PSP94 was located primarily in rat prostate. Furthermore, PSP94 is present at different levels in different lobes of rat prostate, with significant levels detectable only in the lateral lobe (LP). In addition, the most abundant PSP94 expression was found in the prostate lobe secretions, and PSP94 levels in LP secretions were at least seven times higher than in secretions from the dorsal prostate (DP). The rat ventral prostate (VP) and other regions of the male accessory glands were found to be almost completely devoid of PSP94. Since most rat prostate dysplasia induced by steroid hormone treatment occurs only in dorsolateral prostate, prostate tissue-specific expression and the expression of PSP94 in dorsolateral, but not other, lobes of the prostate suggest a potential role in prostate targeting and prostate cancer development.     

The Evaluation of Protein Structure Prediction Results

Methods for protein structure prediction are flourishing and becoming widely available to both experimentalists and computational biologists. However, how good are they? What is their range of applicability and how can we know which method is better suited for the task at hand? These are the questions that this review tries to address, by describing the worldwide Critical Assessment of techniques for protein Structure Prediction (CASP) initiative and focusing on the specific problems of assessing the quality of a protein 3D model.     

Levels of superoxide dismutase and malondialdehyde in primary spontaneous pneumothorax

The aim of the present study is to determine whether patients with primary spontaneous pneumothorax (PSP) are subject to oxidative stress. For this purpose, we measured the activities of red blood cell superoxide dismutase, which is an antioxidant enzyme, and the level of plasma malondialdehyde, which is one of the lipid peroxidation markers, in a group of patients with PSP. The study was carried out with 16 patients with PSP and 24 healthy individuals. The two groups were similar to each other in terms of sex, age and smoking attitudes. Erythrocyte superoxide dismutase activity was found to be significantly lower in patients with PSP than in the control group (p < 0.01). The plasma malondialdehyde levels were significantly high in patients with PSP (p < 0.01). Our results suggest that oxidative stress may contribute to the pathogenesis of PSP.     

Plasmatocyte-spreading peptide influences hemocyte behavior via eicosanoids

Hemocyte-spreading behavior is required for expressing a cellular immune response, nodulation, which clears the vast majority of invading microbes from circulation. The nodulation response is completed by a layer of plasmatocytes, which spread over the nodule and initiate a malanization process leading to darkened nodules. Plasmatocyte-spreading peptide (PSP), the first reported insect cytokine, is responsible for mediating the spreading and attachment of some subclasses of plasmatocytes to nodules. Prostaglandins (PGs), one group of eicosanoids formed from arachidonic acid (AA), also mediate plasmatocyte spreading (PS), although the potential interactions between the PSP and PG signal transduction pathways have not been investigated. We tested our hypothesis that PSP acts via biosynthesis of eicosanoids, specifically PGs, in the beet armyworm, Spodoptera exigua. In this study, we report that (1) PSP and PGE(2) independently stimulated Ca(++)-dependent PS, (2) inhibitors of PG biosynthesis reversibly blocked PS, (3) dsRNA silencing the gene encoding proPSP blocked PS, which was rescued by PSP and by AA, (4) PSP-stimulated PS was reversibly impaired by inhibitors of PG biosynthesis, and (5) the inhibitor-impaired spreading was rescued by AA. Taken together, these points strongly support our model showing that PSP acts via a plasmatocyte-surface receptor, which stimulates biosynthesis of the PGs responsible for mediating plasmatocytes spreading.     

Plasmatocyte spreading peptide induces spreading of plasmatocytes but represses spreading of granulocytes.

In most Lepidoptera, plasmatocytes and granulocytes are the two hemocyte classes capable of adhering to foreign targets. Previously, we identified plasmatocyte spreading peptide (PSP1) from the moth Pseudoplusia includens and reported that it induced plasmatocytes to rapidly spread on foreign surfaces. Here we examine whether the response of plasmatocytes to PSP1 was influenced by cell density or culture conditions, and whether PSP1 affected the adhesive state of granulocytes. Plasmatocyte spreading rates were clearly affected by cell density in the absence of PSP1 but spreading was density independent in the presence of PSP1. PSP1 also induced plasmatocytes in agarose-coated culture wells to form homotypic aggregations rather than spread on the surface of culture wells. In contrast, granulocytes rapidly spread in a density independent manner in the absence of PSP1, but were dose-dependently inhibited from spreading by the addition of peptide. An anti-PSP1 polyclonal antibody neutralized the spreading activity of synthetic PSP1. This antibody also neutralized the plasmatocyte spreading activity of granulocyte-conditioned medium, and significantly delayed plasmatocyte spreading when cells were cultured at a high density in unconditioned medium. These results suggested that the spreading activity derived from granulocytes is due in part to PSP1. Pretreatment of plasmatocytes with trypsin had no effect on PSP1-induced aggregation but PSP1-induced aggregations were readily dissociated by trypsin. This suggested that PSP1 is not an adhesion factor but induces adhesion by stimulating a change in the cell surface of plasmatocytes. Synthetic PSP1 also induced aggregation of plasmatocytes from other Lepidoptera indicating that regulation of hemocyte activity by PSP1-related peptides may be widespread. Arch.     

Effects of pollen load, parasitoids and the environment on pre-dispersal seed predation in the cleistogamous Ruellia nudiflora

Few studies have simultaneously addressed the effects of biotic and abiotic factors on pre-dispersal seed predation (PSP). Plant–seed predator interactions may be influenced by natural enemies and pollinators (the latter through changes in fruit or seed traits), and the activity of pre-dispersal seed predators and their natural enemies may both be affected by the abiotic environment. Additionally, in the case of cleistogamous plants with fruit dimorphism, PSP may be biased towards larger and more seeded chasmogamous (CH) fruits [relative to the smaller cleistogamous (CL) fruits], and the effects of biotic and abiotic factors may be contingent upon this fruit dimorphism. We studied PSP in the cleistogamous Ruellia nudiflora using a split-plot experimental design and asked the following: (1) is PSP biased towards CH fruits and is there an effect of pollen load on PSP? (2) Do parasitoids influence PSP and is their effect influenced by pollen load or fruit type? And (3) do light and water availability modify PSP and parasitoid effects? PSP was higher for CH relative to CL fruits, and under low water availability it was lower for pollen-supplemented CH fruits relative to open-pollinated CH fruits. Parasitoids were not influenced by abiotic conditions, but their negative effect on PSP was stronger for pollen-supplemented CH fruits. Overall, we show that fruit dimorphism, abiotic factors and natural enemies affect PSP, and that these effects can be non-additive.     

HIV Status Awareness,Partnership Dissolution and HIV Transmission in Generalized Epidemics

Objectives

HIV status aware couples with at least one HIV positive partner are characterized by high separation and divorce rates. This phenomenon is often described as a corollary of couples HIV Testing and Counseling (HTC) that ought to be minimized. In this contribution, we demonstrate the implications of partnership dissolution in serodiscordant couples for the propagation of HIV.

Methods

We develop a compartmental model to study epidemic outcomes of elevated partnership dissolution rates in serodiscordant couples and parameterize it with estimates from population-based data (Rakai, Uganda).

Results

Via its effect on partnership dissolution, every percentage point increase in HIV status awareness reduces HIV incidence in monogamous populations by 0.27 percent for women and 0.63 percent for men. These effects are even larger when the assumption of monogamy can be relaxed, but are moderated by other behavior changes (e.g., increased condom use) in HIV status aware serodiscordant partnerships. When these behavior changes are taken into account, each percentage point increase in HIV status awareness reduces HIV incidence by 0.13 and 0.32 percent for women and men, respectively (assuming monogamy). The partnership dissolution effect exists because it decreases the fraction of serodiscordant couples in the population and prolongs the time that individuals spend outside partnerships.

Conclusion

Our model predicts that elevated partnership dissolution rates in HIV status aware serodiscordant couples reduce the spread of HIV. As a consequence, the full impact of couples HTC for HIV prevention is probably larger than recognized to date. Particularly high partnership dissolution rates in female positive serodiscordant couples contribute to the gender imbalance in HIV infections.     

Psychrophilic trypsin-type protease from Serratia proteamaculans

A preparative method for purification of a novel protease from the psychrotolerant Gram-negative microorganism Serratia proteamaculans (PSP) was developed using affinity chromatography on BPTI-Sepharose. It yielded electrophoretically homogeneous PSP preparation of 60 kD. The PSP properties (temperature and pH stability, high catalytic efficiency) indicate that this enzyme can be defined as a psychrophilic protease. Inhibitory analysis together with substrate specificity indicates that the studied PSP exhibits properties of serine trypsin-like and Zn-dependent protease.