Tuning of the optical and electrochemical properties of the primary donor bacteriochlorophylls in the reaction centre from Rhodobacter sphaeroides: spectroscopy and structure

A series of mutations have been introduced at residue 168 of the L-subunit of the reaction centre from Rhodobacter sphaeroides. In the wild-type reaction centre, residue His L168 donates a strong hydrogen bond to the acetyl carbonyl group of one of the pair of bacteriochlorophylls (BChl) that constitutes the primary donor of electrons. Mutation of His L168 to Phe or Leu causes a large decrease in the mid-point redox potential of the primary electron donor, consistent with removal of this strong hydrogen bond. Mutations to Lys, Asp and Arg cause smaller decreases in redox potential, indicative of the presence of weak hydrogen bond and/or an electrostatic effect of the polar residue. A spectroscopic analysis of the mutant complexes suggests that replacement of the wild-type His residue causes a decrease in the strength of the coupling between the two primary donor bacteriochlorophylls. The X-ray crystal structure of the mutant in which His L168 has been replaced by Phe (HL168F) was determined to a resolution of 2.5 A, and the structural model of the HL168F mutant was compared with that of the wild-type complex. The mutation causes a shift in the position of the primary donor bacteriochlorophyll that is adjacent to residue L168, and also affects the conformation of the acetyl carbonyl group of this bacteriochlorophyll. This conformational change constitutes an approximately 27 degrees through-plane rotation, rather than the large into-plane rotation that has been widely discussed in the context of the HL168F mutation. The possible structural basis of the altered spectroscopic properties of the HL168F mutant reaction centre is discussed, as is the relevance of the X-ray crystal structure of the HL168F mutant to the possible structures of the remaining mutant complexes.     

Protein-lipid interactions in the purple bacterial reaction centre

The purple bacterial reaction centre uses the energy of sunlight to power energy-requiring reactions such as the synthesis of ATP. During the last 20 years, a combination of X-ray crystallography, spectroscopy and mutagenesis has provided a detailed insight into the mechanism of light energy transduction in the bacterial reaction centre. In recent years, structural techniques including X-ray crystallography and neutron scattering have also been used to examine the environment of the reaction centre. This mini-review focuses on recent studies of the surface of the reaction centre, and briefly discusses the importance of the specific protein-lipid interactions that have been resolved for integral membrane proteins.     

Morphometric classification of hairy cell leukemia in bone marrow trephine biopsy

OBJECTIVE: To analyze cytologic and histologic parameters in bone marrow trephine biopsy in an attempt to define heterogeneity of hairy cell leukemia cells. STUDY DESIGN: The study group consisted of 28 trephine biopsies. Immunohistochemistry for CD20 antigen was used. Image processing and measurements were performed with AnalySIS 3.0 image analysis system (Soft Imaging System GmbH, Germany) and custom built programs. For planimetric measurements of nuclei, automatic segmentation was implemented. The measured parameters were: surface area, perimeter, minimum, mean and maximum diameter, and a set of form factors. Relative volumes of bone trabeculae, adipose tissue, hematopoietic tissue and neoplastic infiltrate were assessed by the point counting method. Nuclear volume was measured by the point sampled intercept method. Bone marrow fibrosis was assessed using a curvilinear line test system. RESULTS: Significant variability of cell nuclei was found, and their classification into 3 types was possible. The relative frequency of those types was different in various cases and allowed subdivision of cases into 3 groups that differed in some clinical and histologic manifestations. CONCLUSION: The present study demonstrated the heterogeneity of cell populations of hairy cell leukemia.     

Melatonin concentrations in patients with large goiter before and after surgery

OBJECTIVES: Surgical removal of a very large goiter may traumatize adjacent anatomical structures. The manipulations that involve superior cervical ganglia may alter melatonin secretion. To test this hypothesis we decided to study diurnal serum melatonin profiles in patients with a very large goiter before and after the surgery. MATERIAL AND METHODS:The study was performed on 10 women (mean age-46.5+/-1.6 years; mean+/-SEM; range 39-54 years) with very large non-toxic nodular goiter (mean thyroid volume-125.8+/-25.9 cm (3); mean+/-SEM; range 82.6-326.7 cm(3)). Diurnal serum melatonin profiles were estimated two days before the operation and 10 days after the surgery. Blood samples were collected at 08:00, 12:00, 16:00, 20:00, 22:00, 24:00, 02:00, 04:00, 06:00 and 08:00 h. Melatonin concentration was measured using RIA kit. RESULTS: Nocturnal serum melatonin concentrations (at 24, 02, and 04 hours) were significantly higher after the surgery than before the operation. CONCLUSIONS: Very large goiter may compress the superior cervical ganglia altering indirectly the melatonin synthesis. It cannot be excluded, however, that the presence of the large goiter in some other way affects melatonin secretion.     

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

The α_1A-AR is thought to couple predominantly to the Gα_q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gα_q coupling-defective variants of α_1A-AR, as well as a combination of Ca²⁺ channel blockers, uncovered cross-talk between α_1A-AR and β₂-AR that leads to potentiation of a Gα_q-independent signaling cascade in response to α_1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α_1A-AR. α_1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α_1A-AR. Iso induced signaling at α_1A-AR was further interrogated by probing steps along the Gα_q /PLC, Gα_s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α_1A-AR, and CHO_α_1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca²⁺ mobilization that could be blocked by α_1A-AR selective antagonists. The kinetics of Iso induced Ca²⁺ transients differed from typical Gα_q- mediated Ca²⁺ mobilization, lacking both the fast IP₃R mediated response and the sustained phase of Ca²⁺ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α_1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gα_q.     

Substance P and Chronic Pain in Patients with Chronic Inflammation of Connective Tissue

Objective

Evidence suggests that substance P (SP) is involved in chronic joint inflammation, such as the pathogenesis of rheumatoid arthritis and osteoarthritis. The goal of the research was to evaluate the correlation between chronic pain and changes in the SP level in patients with chronic inflammation of the connective tissue.

Methods

Patients with osteoarthritis and rheumatoid arthritis were enrolled in this study. The relationship between chronic pain intensity and the serum SP concentration was evaluated in these groups of patients with osteoarthritis and rheumatoid arthritis.

Results

The results showed a positive correlation between the serum SP concentrations and chronic pain intensity.

Conclusions

1. The SP serum concentration was significantly different between the groups of patients with OA and RA. 2. There was a positive correlation between the serum SP concentration and chronic pain intensity in OA and RA patients.     

Erythrocyte-associated transients in capillary PO2: an isovolemic hemodilution study in the rat spinotrapezius muscle

Mathematical simulations of oxygen delivery to tissue from capillaries that take into account the particulate nature of blood flow predict the existence of oxygen tension (Po(2)) gradients between erythrocytes (RBCs). As RBCs and plasma alternately pass an observation point, these gradients are manifested as rapid fluctuations in Po(2), also known as erythrocyte-associated transients (EATs). The impact of hemodilution on EATs and oxygen delivery at the capillary level of the microcirculation has yet to be elucidated. Therefore, in the present study, phosphorescence quenching microscopy was used to measure EATs and Po(2) in capillaries of the rat spinotrapezius muscle at the following systemic hematocrits (Hct(sys)): normal (39%) and after moderate (HES1; 27%) or severe (HES2; 15%) isovolemic hemodilution using a 6% hetastarch solution. A 532-nm laser, generating 10-micros pulses concentrated onto a 0.9-microm spot, was used to obtain plasma Po(2) values 100 times/s at points along surface capillaries of the muscle. Mean capillary Po(2) (Pc(O(2)); means +/- SE) significantly decreased between conditions (normal: 56 +/- 2 mmHg, n = 45; HES1: 47 +/- 2 mmHg, n = 62; HES2: 27 +/- 2 mmHg, n = 52, where n = capillary number). In addition, the magnitude of Po(2) transients (DeltaPo(2)) significantly decreased with hemodilution (normal: 19 +/- 1 mmHg, n = 45; HES1: 11 +/- 1 mmHg, n = 62; HES2: 6 +/- 1 mmHg, n = 52). Results suggest that the decrease in Pc(O(2)) and DeltaPo(2) with hemodilution is primarily dependent on Hct(sys) and subsequent microvascular compensations.     

Contributions of collision rate and collision efficiency to erythrocyte aggregation in postcapillary venules at low flow rates

Red blood cell aggregation at low flow rates increases venous vascular resistance, but the process of aggregate formation in these vessels is not well understood. We previously reported that aggregate formation in postcapillary venules of the rat spinotrapezius muscle mainly occurs in a middle region between 15 and 30 microm downstream from the entrance. In light of the findings in that study, the main purpose of this study was to test two hypotheses by measuring collision frequency along the length of the venules during low flow. We tested the hypothesis that aggregation rarely occurs in the initial 15-microm region of the venule because collision frequency is very low. We found that collision frequency was lower than in other regions, but collision efficiency (the ratio of aggregate formation to collisions) was almost nil in this region, most likely because of entrance effects and time required for aggregation. Radial migration of red blood cells and Dextran 500 had no effect on collision frequency. We also tested the hypothesis that aggregation was reduced in the distal venule region because of the low aggregability of remaining nonaggregated cells. Our findings support this hypothesis, since a simple model based on the ratio of aggregatable to nonaggregatable red blood cells predicts the time course of collision efficiency in this region. Collision efficiency averaged 18% overall but varied from 0 to 52% and was highest in the middle region. We conclude that while collision frequency influences red blood cell aggregate formation in postcapillary venules, collision efficiency is more important.     

Peptides derived from type I thrombospondin repeat-containing proteins of the CCN family inhibit proliferation and migration of endothelial cells

Angiogenesis, or neovascularization, is tightly orchestrated by endogenous regulators that promote or inhibit the process. The fine-tuning of these pro- and anti-angiogenic elements (the angiogenic balance) helps establish the homeostasis in tissues, and any aberration leads to pathologic conditions. The type I thrombospondin repeats are a family of protein structural elements involved in the control of angiogenesis, and some proteins containing these repeats have been identified as negative regulators of angiogenesis. Here we identify a set of 11 novel, anti-angiogenic 18–20-amino acid peptides that are derived from proteins that belong to the CCN protein family and contain type I thrombospondin motifs. We have named these peptides spondinstatin-1, cyrostatin, connectostatin, nephroblastostatin, wispostatin-2, wispostatin-3, netrinstatin-5C, netrinstatin-5D, adamtsostatin-like-4, fibulostatin-6.1, and complestatin-C6 to reflect their origin. We have shown that these peptides inhibit proliferation and migration of human umbilical vein endothelial cells in vitro. By conducting a clustering analysis of the amino acid sequences using sequence similarity criteria and of the experimental results using a hierarchical clustering algorithm, we have demonstrated that there is an underlying correlation between the sequence and activity of the identified peptides. This combination of experimental and computational approaches introduces a novel systematic framework for studying peptide activity, identifying novel peptides with anti-angiogenic activity, and designing mimetic peptides with tailored properties.