Electron crystallography of macromolecular periodic arrays on phospholipid monolayers

Electron crystallography has the potential of yielding structural information equivalent to x-ray diffraction. The major difficulty has been preparing specimens with the required structural order and size for diffraction and imaging in the electron microscope. 2D crystallization on phospholipid monolayers is capable of fulfilling both of these requirements. Crystals can form as a result of specific interactions with a protein's ligand or an analog, suitably linked to a lipid tail; or on a surface of complementary head-group charge. With such choices, the availability of a suitable lipid is limited only by synthetic chemistry. Ultimately, it is the quality and regularity of the protein-protein interactions that determine the crystalline order, as it is with any protein crystal. In the case of streptavidin, the monolayer crystal diffracts beyond 2.5 Å. A 3 Å projection map reconstructed from electron diffraction amplitudes and phases from images shows density which can be interpreted as β-sheets and clusters of side chains. It remains to be shown that the monolayer crystals are flat and diffract as well at high tilt angle as untilted. Technological issues such as charging must be resolved. With parallel advances in data collection and processing, electron crystallography of monolayer macromolecular crystals will eventually take its place beside x-ray crystallography and NMR as a routine and efficient structural technique.     

Adrenomedullin is increased by pulsatile shear stress on the vascular endothelium via periodic acceleration (pGz)

Periodic acceleration (pGz) is produced by a platform which moves the supine body repetitively in a headward to footward direction. The imparted motion produces pulsatile shear stress on the vascular endothelium. Pulsatile shear stress on the vascular endothelium has been shown to elicit production of a host of cardioprotective, cytoprotective mediators. The purpose of this study was to ascertain if pGz also enhances production of adrenomedullin (AM) in normal healthy swine. Twelve pigs (weight range 20-30 kg) were anesthetized, intubated and placed on conventional mechanical ventilation. All animals were secured to the motion platform. In one group (pGz) (n=7) was activated for 1h, and monitored for an additional 3h. A control group (CONT) (n=5) served as time control. Arterial blood gases, hemodynamic measurements, and serum for AM, interleukin 4, 6 and thromboxane B(2) (TBXB2) were measured at baseline, immediately after pGz, and 3h after pGz had been discontinued. There was no significant change from baseline value in IL-4, IL-6 or TBXB2. Mean arterial blood pressure decreased in pGz-treated animals from 115+/-10 at baseline to 90+/-8 after 60 min of pGz (p<0.01). AM levels increase from 776+/-176 pg/ml baseline to 1160+/-68 pg/ml immediately after pGz, and remained elevated to 1584+/-160 pg/ml, 3h after pGz (p<0.01 vs. BL). This is the first report of AM-enhanced production using a non-invasive method of increasing pulsatile shear stress on the vascular endothelium. pGz increases production of AM in normal healthy swine. These changes are independent of IL-4, IL-6 or TBXB2 production.     

Gallium-transferrin as a macromolecular tracer of vascular permeability

Labeling of plasma transferrin with gallium was investigated to determine whether the gallium-transferrin complex could be effectively used as a macromolecular tracer in studies of capillary permeability using Positron Emission Tomography (PET). Three gallium-plasma preparations were tested and 2 h bio-distribution studies were performed in rats. The three preparations gave similar blood clearance and tissue distribution data, but the methods used for evaluating gallium-transferrin binding were found to be suboptimal. Gallium clearance from blood was biexponential with both components faster than that of ¹²⁵I-albumin. Gallium distribution spaces in all tissues including intracerebral Walker-256 tumors were larger than those of albumin. These results indicate a relative instability of the gallium-transferrin complex in vivo, which appears to preclude its use as an acceptable radiolabeled protein for vascular permeability studies using PET.     

Effect of light intensity on macromolecular synthesis in cyanobacteria

The light-dependent incorporation of NaH¹⁴CO₃ into low molecular weight compounds, polysaccharide, or protein was determined in cultures of the cyanobacteriumMerismopedia tenuissima incubated at a series of light intensities. There was an inverse relationship between incorporation into polysaccharide and protein. At light intensities of 90 E/m²/sec or above, relative incorporation of radioisotope into polysaccharide was greatest and relative incorporation into protein was lowest. Optimal relative protein accumulation occurred in samples incubated at 20 E/m²/sec. A broader optimum of light intensity for maximal protein accumulation was found if ammonia rather than nitrate was the nitrogen source. Physiological adaptation of cultures to growth at a particular light intensity did not alter the pattern of macromolecular incorporation when those cultures were tested over the series of light intensities. The response of cultures ofOscillatoria rubescens to light intensity was similar to that ofM. tenuissima, although incorporation into low molecular weight compounds was significantly greater.The effect of light intensity on macromolecular synthesis in a natural population ofOscillatoria rubescens was also determined. A pattern similar to that observed in batch cultures ofO. rubescens was occasionally found, but in other experiments there was no increase in relative protein incorporation when light intensity was decreased.     

The uptake of macromolecular materials in the hindgut of viviparous rockfish embryos

Regions of the gut of near-term embryos and new-born larvae of the viviparous rockfish Sebastes taczanowskii were examined histologically and immunohistochemically in order to ascertain whether they possessed the potential to absorb intact macromolecules. Compared with the hindgut of larvae 24 h after parturition, that of near-term embryos (stage 31) was found to be markedly hypertrophied and to react positively when tested for the presence of eosinophilic materials and with antibodies raised against extracts of the rockfish yolk (ab.a-E). In addition, near-term embryos incubated in Ringer solution containing 1% bovine serum albumin appeared to accumulate the protein within the epithelium of the hindgut. Furthermore, as confirmed by immunodiffusion analysis, the ovarian fluid contained materials which reacted positively to ab.a-E. These results suggest that ingestion of intact macromolecules occurs during the late embryonic stages and that the yolk-like substances detected within the hindgut epithelium of such embryos may be derived from the bathing ovarian fluid.     

Early vascular alterations in the diabetic rat heart

The aim of this study was to evaluate whether there are vascular alterations in diabetes prior to atherosclerotic lesions. Four and 12 weeks after induction of diabetes (streptozotocin, 60 mg/kg b.w.) in rats characterized by low plasma insulin, hyperglycaemia, glucosuria, and loss of body weight, however, without morphological alterations of the cardiac vasculature--local myocardial perfusion parameters were estimated in the epicardium of Langendorff perfused hearts using fluorescence indicator (FITC-dextrane, MG 3kD) washout kinetics after bolus injection. The elution profile was characterized by a fast vascular and a slow transendothelial component. In diabetic hearts, vascular as well as transendothelial washout were prolonged. Concomitantly, the vascular perfusion volume and the amount of indicator exchanged with the interstitium was significantly diminished. Vascular response to bradykinin (5 nmol/l) was moderately attenuated and the limitations in transendothelial indicator exchange could not be affected by bradykinin, although vascular perfusion volume was significantly increased. These findings clearly indicate disturbed local myocardial perfusion early in the development of diabetes.     

Vanadate-resistant mutants of Candida albicans show alterations in phosphate uptake

Phosphate uptake studies in different strains of the dimorphic pathogenic yeast Candida albicans were undertaken to show that this yeast actively transported phosphate with an apparent Km in the range of 90-170 microM. The uptake was pH dependent and derepressible under phosphate starvation. Vanadate-resistant (van) mutants of C. albicans showed a 20-70% reduction in the rate of phosphate uptake in high phosphate medium and was associated with an increased Km and reduced Vmax. The magnitude of derepression under phosphate starvation was different between van mutants. These results demonstrate that van mutants may have developed resistance by modifying the rate of entry of vanadate.     

Ca handling alterations and vascular dysfunction in diabetes

More than 65% of patients with diabetes mellitus die from cardiovascular disease or stroke. Hyperglycemia, due to either reduced insulin secretion or reduced insulin sensitivity, is the hallmark feature of diabetes mellitus. Vascular dysfunction is a distinctive phenotype found in both types of diabetes and could be responsible for the high incidence of stroke, heart attack, and organ damage in diabetic patients. In addition to well-documented endothelial dysfunction, Ca²⁺ handling alterations in vascular smooth muscle cells (VSMCs) play a key role in the development and progression of vascular complications in diabetes. VSMCs provide not only structural integrity to the vessels but also control myogenic arterial tone and systemic blood pressure through global and local Ca²⁺ signaling. The Ca²⁺ signalosome of VSMCs is integrated by an extensive number of Ca²⁺ handling proteins (i.e. channels, pumps, exchangers) and related signal transduction components, whose function is modulated by endothelial effectors. This review summarizes recent findings concerning alterations in endothelium and VSMC Ca²⁺ signaling proteins that may contribute to the vascular dysfunction found in the diabetic condition.     

Effect of polyamines on the fidelity of macromolecular synthesis

Addition of polyamines to in vitro systems containing suboptimal concentrations of Mg2+ markedly stimulated protein and RNA synthesis. This stimulation is observed only within a marrow range of polyamine concentration. The extend of stimulation of RNA synthesis was dependent on assay conditions. Addition of spermidine to the wheat germ system not only stimulated poly(U) directed polyphenylalanine synthesis, but also reduced the misincorporation of leucine. MS2-coat protein synthesis, studied in an E.coli cell-free system using either one of the two glutamyl-tRNAs as the only source of glutamine, suggested that in the presence of spermidine, codon recognition by these two isoacceptor tRNA molecules was more stringent. From these results it is concluded that polyamines contribute to the specificity of codon/anticodon interactions and thereby increase the fidelity of protein synthesis.     

Effect of mixed macromolecular crowding agents on protein folding

In cells, proteins fold and unfold in the presence of macromolecules with various sizes and shapes. Recent experiments by Liang and coworkers (J Biol Chem 2004;279:55109-55116; J Mol Biol 2006;364:469-482) show that protein refolding is enhanced by a mixture of two different crowding agents relative to the individual crowding agents and an optimal mixing ratio exists. Here, we present a theory that predicts the existence of an optimal mixing ratio. The theory is based on models for calculating the changes in the chemical potentials of the folded and unfolded states by a mixture of crowders. The existence of an optimal mixing ratio results from the dependences of these chemical-potential changes on crowder sizes and concentrations, which can be argued to be quite general. We further predict that, for any crowding agent, the stabilizing effect can be optimized both by varying the molecular weight and the mixing ratio of two species with different molecular weights.     

Correlation among shear rate measures in vascular flows

A variety of shear rate measures have been calculated from hemodynamic data obtained by laser Doppler anemometry in flow-through casts of human aortic bifurcations. Included are measures sensitive to the mean and amplitude of the shear rate, its maximum rate of change, the duration of stasis and flow reversal near the wall, and the unidirectionality of the flow. Many of these measures are highly correlated with one another. This suggests that that it will be difficult to identify from in vivo measurements those aspects of the flow field to which the vessel wall is most sensitive. It may be possible to separate the effects of purely temporal factors (e.g., the duration of flow reversal) from those related to wall shear stress.     

Effect of temperature on growth and macromolecular biosynthesis in cryptococcus species

Cryptococcus neoformans, a pathogenic yeast, grows at temperatures between 25 and 37°C. However, the closely related non-pathogen C. albidus exhibits restricted growth at temperatures above ambient with little or no growth at 37°C. The inhibition of growth of the non-pathogen, as measured by turbidity, cell number, and per cent budding, is reversible after 48 hr at the non-permissive temperature (37°C). Growth cessation at 37°C is accompanied by a corresponding decrease in DNA synthesis, which is not observed in C. neoformans. RNA and protein synthesis in C. albidus and C. neoformans are only slightly affected at the elevated temperature. Degradation by nucleases does not seem to account for the differences found in this cumulative DNA synthesis in C. albidus at 25 and 37°C. These facts suggest that C. albidus may possess a thermo-sensitive defect in the machinery responsible for the initiation of DNA replication.     

Effect of short-chain organic acids on macromolecular synthesis in Escherichia coli

Incubating cultures of Escherichia coli with propionic acid (5 mmol/l) or formic acid (10 mmol/l) at pH 5.0 produced bacteriostasis lasting 30 and 120 min respectively. During this time rates of RNA, DNA, protein, lipid and cell wall synthesis were reduced. Growth resumed after continued incubation in the presence of acid, but cells from acid-treated cultures were larger than controls. DNA synthesis was particularly sensitive to the presence of the propionic or formic acid.     

Effect of high shear on proteins

Shear is present in almost all bioprocesses and high shear is associated with processes involving agitation and emulsification. The purpose of this study is to investigate the effect of high shear and high shear rate on proteins. Two concentric cylinder-based shear systems were used. One was a closed concentric-cylinder shear device (CCSD) and the other was a homogenizer with a rotor/stator assembly. Mathematical modeling of these systems allowed calculation of the shear rate and shear. The CCSD generated low shear rates (a few hundred s(-1)), whereas the homogenizer could generate very high shear rates (> 10(5) s(-1)). High shear could be achieved in both systems by increasing the processing time. Recombinant human growth hormone (rhGH) and recombinant human deoxyribonuclease (rhDNase) were used as the model proteins in this study. It was found that neither high shear nor high shear rate had a significant effect on protein aggregation. However, a lower melting temperature and enthalpy were detected for highly sheared rhGH by using scanning microcalorimetry, presumably due to some changes in protein's conformation. Also, SDS-PAGE indicated the presence of low molecular-weight fragments, suggesting that peptide bond breakage occurred due to high shear. rhDNase was relatively more stable than rhGH under high shear. No conformational changes and protein fragments were observed. (c) 1996 John Wiley & Sons, Inc.     

Sexual dimorphism in angiotensin II-induced hypertension and vascular alterations

Sex differences in the degree of high blood pressure have been described in several forms of experimental animal models of hypertension. However, the influence of sex on angiotensin II-induced hypertension has not been studied. In the present study, we investigated and compared the effects of chronic angiotensin II treatment on blood pressure and vascular function in male and female rats. Chronic treatment with angiotensin II (0.7 mg/kg daily for 10 d) significantly raised arterial blood pressure in male but not female Sprague-Dawley rats; it upregulated the NAD(P)H oxidase gp67 phox subunit in the aorta of male but not female rats; and it exaggerated the vasoconstrictor responses to norepinephrine and serotonin in the mesenteric vascular bed (MVB) of male but not female rats. Vasodilator responses to acetylcholine (ACh) but not papaverine (PPV) or isoprenaline (ISO) were reduced in the MVB of angiotensin II-treated male but not female rats. ACh, but not PPV or ISO dilatory responses were potentiated in the MVB of angiotensin II-treated female rats. The present findings demonstrate that exogenous angiotensin II upregulates aortic NAD(P)H oxidase gp67 phox subunit, and induces hypertension and mesenteric vascular dysfunction only in male rats.