Simulating electrostatic energies in proteins: perspectives and some recent studies of pKas, redox, and other crucial functional properties

Electrostatic energies provide what is arguably the most effective tool for structure-function correlation of biological molecules. Here, we provide an overview of the current state-of-the-art simulations of electrostatic energies in macromolecules, emphasizing the microscopic perspective but also relating it to macroscopic approaches. We comment on the convergence issue and other problems of the microscopic models and the ways of keeping the microscopic physics while moving to semi-macroscopic directions. We discuss the nature of the protein dielectric "constants" reiterating our long-standing point that the dielectric "constants" in semi-macroscopic models depend on the definition and the specific treatment. The advances and the challenges in the field are illustrated considering different functional properties including pK(a)'s, redox potentials, ion and proton channels, enzyme catalysis, ligand binding, and protein stability. We emphasize the microscopic overcharging approach for studying pK(a) 's of internal groups in proteins and give a demonstration of power of this approach. We also emphasize recent advances in coarse grained models with a physically based electrostatic treatment and provide some examples including further directions in treating voltage activated ion channels.     

Ultrastructural study of cardiotoxicity and skin alterations in the golden hamster after treatment with 8 different anthracyclines]

Golden hamsters were submitted to i.p. administration during 4 weeks of 8 anthracyclines, adriamycin (ADM), detorubicin (DTR), daunorubicin (DNR), 4'-epi-adriamycin (eADM), adriamycin hydrochloride (ADMh), rubidazon (RBZ), aclacinomycin (ACM) and AD32, at doses equivalent to 3/4 of those which are optimally oncostatic on murine L1210 leukemia. The comparative study of the mortality, the electron microscopic (EM) alterations of the myocardium, and the light microscopic (EM) alterations of the myocardium, and the light microscopic (LM) lesions of the skin, show that ACM and AD32 are the least toxic drugs. EM detected almost no early lesions of myocardium in ACM treated animals, but, after 4 week's treatment, severe cardiac alterations appeared which, like those after AD32 treatment, are non lethal and reversible. Similarly. LM revealed no histologic changes in the skin following ACM and AD32 administrations, but pathologic alterations, atrophy and alopecia, were observed in animals receiving all other drugs.     

The filamentous morphotype Eikelboom Type 1863 is not a single genetic entity

Five isolates of a filamentous bacterial morphotype with the distinctive diagnostic microscopic features of Eikelboom Type 1863 were obtained from activated sludge sewage treatment plants in Victoria, Australia. On the basis of phenotypic evidence and 16S rDNA sequence data, these isolates proved to be polyphyletic. Two (Ben 06 and Ben 06C) are from the Chryseobacterium subgroup which is in the Cytophaga group, subdivision I of the Flexibacter – Cytophaga – Bacteroides phylum. Two (Ben 56 and Ben 59) belong to the genus Acinetobacter , and one (Ben 58) is a Moraxella sp., closest to Mor. osloensis . The significance of these findings to the reliance on microscopic features for identification of these filamentous bacteria in activated sludge is discussed.     

Experimenteller Beitrag zur Kenntnis der biogenen Amine im Glomus caroticum des Kaninchens

Zusammenfassung Der Einfluß von p-Chlorphenylalanin-methylester-hydrochlorid (PCPA) und Reserpin auf die biogenen Amine des Glomus caroticum von Kaninchen wurde ultrastrukturell und fluoreszenzmikroskopisch untersucht. Die elektronenmikroskopische Analyse ergab keine eindeutigen Kriterien für arzneimittelinduzierte Veränderungen. Fluoreszenzmikroskopisch ließ sich nach Applikation von Reserpin eine ausgeprägte Senkung des Catecholamin- und Indolamin-Gehaltes und nach PCPA eine Abnahme des Serotonins erkennen.

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Electron microscopic and fluorescence microscopic studies of biogenic amines in the rabbit glomus caroticum after treatment with reserpine and PCPA

Summary The effects of p-chlorophenylalanine-methylester-hydrochloride (PCPA) and reserpine on biogenic amines of the rabbit carotid body were investigated ultrastructurally and by fluorescence microscopy. The electron microscopic analysis did not indicate significantly that structural changes result from treatment with reserpine or PCPA. Fluorescence microscopy indicated that PCPA lowered serotonin, and reserpine lowered both catecholamines and indolamines.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Efficacious cellular codelivery of doxorubicin and EGFP siRNA mediated by the composition of PLGA and PEI protected gold nanoparticles

This study reports the simultaneous delivery of EGFP siRNA and the chemotherapeutic drug, doxorubicin by means of the composition that results from the electrostatic interaction between positively charged siRNA-complexes of gold nanoparticles (AuNPs) capped with PEI, 25 kDa (P25-AuNPs) and negatively charged carboxymethyl cellulose formulated PLGA nanoparticles loaded with doxorubicin. The nanoparticles and their facile interaction were studied by means of dynamic light scattering (DLS), zeta potential, transmission electron microscopic (TEM) measurements. The flow cytometric and confocal microscopic analysis evidenced the simultaneous internalization of both labelled siRNA and doxorubin into around 55% of the HeLa cancer cell population. Fluorescence microscopic studies enabled the visual analysis of EGFP expressing HeLa cells which suggested that the composition mediated codelivery resulted in a substantial downregulation of EGFP expression and intracellular accumulation of doxorubicin. Interestingly, codelivery treatment resulted in an increased cellular delivery of doxorubicin when compared to PLGA-DOX alone treatment. On the other hand, the activity of siRNA complexes of PEI-AuNPs was completely retained even when they were part of composition. The results suggest that this formulation can serve as promising tool for delivery applications in combinatorial anticancer therapy.     

Silver amplification of mercury sulfide and selenide: a histochemical method for light and electron microscopic localization of mercury in tissue

A method for light and electron microscopic demonstration of mercury sulfides and mercury selenides in mammalian tissue is presented. Silver ions adhering to the surface of submicroscopic traces of mercury sulfides or selenides in the tissue are reduced to metallic silver by hydroquinone. Physical development thereupon renders deposits of mercury sulfides or mercury selenide visible as spheres of solid silver. Examples of localization of mercury in the central nervous system and various organs from animals exposed to mercury chloride or methyl mercury chloride with or without additional sodium selenide treatment are presented. Selenium treatment results in a considerable increase in the amount of mercury that can be made visible by silver amplification. After mercury chloride treatment, most of the mercury is localized in lysosomes and is only rarely seen in secretory granules. After simultaneous selenium treatment, mercury is also found in nuclei of proximal tubule cells in the kidney and in macrophages. The "sulfide-osmium" method for ultrastructural localization of mercury suggested by Silberberg, Lawrence, and Leider (Arch Environ Health 19:7, 1969) and the light microscopic method using a photographic emulsion suggested by Umeda, Saito, and Saito (Jpn J Exp Med 39:17, 1969) have been experimentally analyzed and commented on.     

Application of Serial Section Method to Determine the Radiotherapy Target Volume for Esophageal Squamous Carcinoma

The three-dimensional conformal radiotherapy (3D CRT) plays an important role in the combination treatment of esophageal carcinoma. However, an accurate estimation of the clinical target volume (CTV) of esophageal squamous carcinoma (ESCC) by 3D CRT is still problematic. This study aimed to provide reference values for CTV estimation. The serial section method was applied to observe the range of the microscopic spread proximally and distally from the tumor. Further, relationships between clinicopathological features and the microscopic spread were analyzed. The positive ratio of the proximal microscopic spread was significantly higher than in the distal spread, especially in the specimens sampled 1 and 2 cm away from the tumor (p < 0.05). Probability of infiltration and metastases was still high in the proximal “3 cm” and distal “4 cm” groups, and became much lower in more distant specimens. Further, ESCC tended to exhibit stronger ascending invasion ability. A single factor analysis showed that tumors with the length of longer than 5 cm, poorer differentiation, lymph nodes metastasis, and more aggressive phase had significantly higher microscopic spread ratio (p < 0.05). A multiple factors analysis showed that differentiation degree and tumor length were the major factors affecting the microscopic spread of ESCC. In conclusion, to cover 95 % of the microscopic spread, a proximal margin of 3.0 cm and a distal margin of 4.0 cm are needed. In order to cover 90 %, proximal and distal 3-cm margins are needed. Clinicopathological features of patients can affect the range of the microscopic spread.     

DNA-induced distamycin A fluorescence

The fluorescent properties of the antibiotic distamycin A were investigated in a range of materials including Trypanosoma cruzi epimastigotes, chicken erythrocytes, calf thymus DNA and synthetic polynucleotides using both microscopic and spectroscopic techniques. A bright blue-white fluorescence was observed from kinetoplast DNA and chromatin after treatment with distamycin A under ultraviolet (365 nm) excitation. Considerable enhancement of distamycin A fluorescence (emission peak at 455 nm under 320-340 nm excitation) was found in the presence of DNA and poly(dA-dT).poly(dA-dT). We discuss a possible explanation for this unexpected fluorescent emission, as well as its implications for microscopic and fluorimetric studies.     

The influence of a single ethanol injection on normal thyroid tissue of the rat

Macro- and microscopic changes in the normal thyroid gland of rats, and in the surrounding tissues 2 and 4 weeks after a single intrathyroidal ethanol injection (IEI), together with the influence of such treatment on the function of the recurrent laryngeal nerves and of the parathyroid glands, were assessed. The intraoperative macroscopic evaluation at 2 weeks (20 rats) and 4 weeks (20 rats) after IEI revealed the presence of a scar at the site of the IEI-treated lobe in seven (35%) and six (30%) rats, respectively, and the reduction of lobe dimensions in thirteen (65%) and fourteen (70%) rats, respectively. The microscopic evaluation of the lobe after IEI showed coagulative necrosis, reduction in thyroid follicle volume, disturbance of follicle structure, haemorrhage, haemosiderin deposits, inflammatory infiltration and fibrosis. No microscopic changes were observed in the tissues surrounding the thyroid, nor in the parathyroid glands located extrathyroidally or in the second thyroid lobe. No vocal cord dysfunction or significant changes in serum calcium levels after IEI were detected.     

Catnip,Nepeta cataria, a morphological comparison of mutant and wild type specimens to gain an ethnobotanical perspective

Nepeta cataria L, the catnip plant, is important in the pet industry for cats and as an herbal medicinal treatment for the fevers, diarrhea, insomnia, and lacking menstruation of humans. A natural mutation of N. cataria produced a novel morphology that warranted investigation to determine how the mutation affected the microscopic features, including catnip’s ethnobotanical storehouse of glandular hairs. The morphology, anatomy, and physiology of this mutant are compared to that of the wild type of catnip to document the major differences. The secondary plant metabolites which facilitate catnip’s ethnobotanical uses are stored in microscopic glandular hairs (trichomes). The trichomes on the mutant and wild type catnip leaves were not shown to differ (scanning and transmission electron microscopy). The feliobotany (use by cats) of N. cataria is discussed in relation to catnip trichomes.     

Towards developing a diagnostic regimen for the treatment follow-up of Trypanosoma brucei gambiense

BALB/c mice infected with a high virulent strain of Trypanosoma brucei gambiense IL3707 were treated intraperitoneally (i.p.) with either Melarsoprol (Mel-B) or PSG(+) buffer as controls. The mice were subsequently monitored regularly for parasites by direct microscopic examination of their tail blood or buffy coat and by polymerase chain reaction (PCR). Mel-B was found to be an effective drug for treatment against T.b. gambiense because at the end of the first treatment schedule, all treated mice were negative for parasites even by PCR, while all the control animals were positive. Three of the five Mel-B treated mice, while parasitologically negative, were PCR positive between 53 and 80 days post infection (DPI), indicating that they still harbored an infection. All treated mice were subsequently negative for parasites even by PCR at 88 DPI. A combination of conventional microscopic examination and PCR offers a good prediction of cure following treatment of trypanosomosis.     

DNA-induced distamycin a fluorescence

Summary The fluorescent properties of the antibiotic distamycin A were investigated in a range of materials including Trypanosoma cruzi epimastigotes, chicken erythrocytes, calf thymus DNA and synthetic polynucleotides using both microscopic and spectroscopic techniques. A bright blue-white fluorescence was observed from kinetoplast DNA and chromatin after treatment with distamycin A under ultraviolet (365 nm) excitation. Considerable enhancement of distamycin A fluorescence (emission peak at 455 nm under 320–340 nm excitation) was found in the presence of DNA and poly(dA-dT)·poly(dA-dT). We discuss a possible explanation for this unexpected fluorescent emission, as well as its implications for microscopic and fluorimetric studies.     

BRIEF INCUBATION OF GAMETANGIA-BEARING CAPS IN ANTIBIOTICS ELIMINATES BRANCHING IN PROGENY OF ACETABULARIA ACETABULUM (CHLOROPHYTA)1

Branching of the stalk of Acetabularia acetabulum L. (Silva) was investigated by inbreeding and by a brief treatment of gametangia with a variety of antibiotics. The position of the branch along the stalk varied, implying that branching was not restricted to any one time in development (base is oldest and apex is youngest). The branching phenotype was not inherited in Mendelian fashion. Although three microscopic structures (“bubbles,”“pustules,” and “scars”) occurred on the stalks of cells that had branched, these structures were not statistically correlated with branching in the population (n=699 cells). However, brief treatment of gametangia with a new antibiotic mixture did eliminate all macro- and microscopic structures associated with branching of the stalk in the subsequent generation. We could not fulfill Koch's postulates or provide clear evidence for the pathogenic nature of cell branching. Our brief antibiotic treatment of gametangaa of Acetabularia acetabulum was rapid, had no adverse effects, and virtually eliminated branching (and any potential pathogens) from laboratory cultures in the subsequent generations. Our method allows biochemical and molecular analyses to proceed uncomplicated by the possible presence of other organisms and provides a clean baseline for the future selection of mutations that may induce heritable branching.     

Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: application in PCR

The procedure of obtaining DNA-containing cell envelopes ("micromummies") of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.     

An electron microscopic method for the mapping of proteins attached to nucleic acids.

An electron microscopic method for demonstrating the presence of and mapping the positions of proteins specifically bound to nucleic acids is described. The nucleic acid-protein complex is treated with dinitrofluorobenzene under conditions such that dinitrophenyl (DNP) groups are attached to nucleophilic groups on the protein, with only a low level of random attachment to the nuclei acid. This product is treated with rabbit anti-DNP IgG. The position of the protein-(DNP)n(IgG)m complex on the nucleic acid strand can be observed by electron microscopy by protein free spreading methods and, in many cases, by cytochrome-c spreading. If necessary for visualization by the latter method, the size of the labeled region can be increased by treatment with goat anti-rabbit IgG. High efficiency of electron microscopic labeling is achieved. Examples studied are: the adenovirus-2 DNA terminal protein, a protein covalently bound to SV40 DNA, DNA polymerase I bound to DNA, E. coli RNA polymerase bound to T7 DNA, and proteins UV crosslinked to avian sarcoma virus RNA.     

Studies on Streptococcus pyogenes. III. The effect of trypsin and a cationic detergent on the structure,permeability, and metabolism of the cell

Electron microscopic observations of Streptococcus pyogenes (strain S43, type 6) treated with trypsin and cetyltrimethylammonium bromide (Cetab) are presented. The concentration of trypsin necessary to remove the M protein antigen was shown to bring about a partial digestion of the cells. The cell protoplasm and protoplasmic membrane were affected. No microscopic changes appeared in the cell wall. Cetab did not alter the appearance of the cells. Both trypsin and detergent altered the permeability of the cell so that citrulline and carbamylphosphate were metabolized. Cells exhibiting enzyme activity on these substrates after Cetab treatment were characterized by the release of lysine, glutamic acid, nucleic acid, and other cellular material. Enzymes responsible for the metabolism of arginine, citrulline, and carbamylphosphate were shown to reside in the cell protoplasm. Cells which had lost their viability after Cetab treatment still possessed the ability to utilize the latter substrates. Under similar conditions the metabolism of glucose did not occur. Normal cells were shown to possess the ability to fluoresce in the presence of a dye. The intensity of the fluorescence was reduced by trypsin treatment.     

Investigations on the turnover of adrenocortical mitochondria

Summary The effects of a chronic treatment with angiotensin II (up to 15 consecutive days) on the mitochondria of the rat zona glomerulosa cells were investigated by electron microscopic and stereological methods. Angiotensin induced a significant increase in the volume of the mitochondrial compartment. Up to the 3rd day of treatment this was due only to the hypertrophy of the organelles, and from the 3rd to the 15th day exclusively to mitochondrial proliferation. The hypothesis that angiotensin controls the growth and proliferation of rat zona glomerulosa mitochondria is discussed.     

折流曝气生物滤池中污染物与微生物沿程变化规律

BBAF滤池是一种新型曝气生物滤池,为考察其中污染物与微生物沿程变化规律,分析了不同厚度滤层对有机物、氨氮等污染物的去除效果,MPN法测定了具有代表性滤层厚度处的异养菌和硝化菌数量;通过镜检观察研究了BBAF滤池中微生物种群的沿程分布特点。研究表明:BBAF滤池中COD浓度随滤层厚度增加逐渐降低,前4个单池对COD有很好的去除效果,但对氨氮的去除作用较弱,其中的微生物以异养菌为主;第5、6、7单池中,有机负荷较低,促进了硝化菌增殖,提高了硝化效果。BBAF滤池的各单池之间存在浓度梯度,使不同滤层中形成了微生物优势种属,有利于BBAF滤池达到最佳运行效果。     

Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: Application in PCR

The procedure of obtaining DNA-containing cell envelopes (“micromummies”) of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.     

The relationship between viability and intracellular pH in the yeast Saccharomyces cerevisiae.

The relationship between viability (cell proliferation activity) and intracellular pH in the yeast Saccharomyces cerevisiae was investigated by using cells that had been deactivated by low-temperature storage, ethanol treatment, or heat treatment. The intracellular pH was measured with a microscopic image processor or a spectrofluorophotometer. At first, the intracellular pH measurements of individual cells were compared with slide culture results by microscopic image processing. A clear correlation existed between the proliferation activity and intracellular pH. Moreover, by spectrofluorophotometry analysis, it was found that there was a relationship between the viability and intracellular pH of brewing yeast under conditions of low external pH (n = 15, r = 0.960, P = 0.001). This relationship was also observed in baker's yeast (n = 13, r = 0.950, P = 0.001). On the other hand, when the fluorescein staining method was used in these experiments, the relationship between viability and staining percentage was not observed. From these results, intracellular pH was found to be a sensitive factor for estimating yeast physiology. The possible role of cell deterioration is also discussed.