A dynamic model for the structure of acyl carrier protein in solution

The determination of solution structures of proteins using two-dimensional NMR data is commonly based on the assumption that the structure can be represented by a single rigid conformer. We present here a procedure whereby this assumption can be relaxed and illustrate its application to acyl carrier protein from Escherichia coli, a small negatively charged protein with no internal disulfide bonds. The methodology rests on a model having two distinct conformers in dynamic equilibrium. Use of this two-state model results in a dramatic improvement in fit to cross-relaxation-derived distance constraints and a substantial lowering of molecular mechanics energies for individual conformers of acyl carrier protein. The two-state model retains the three-helix motif previously identified on the basis of a one-state structure, but substantial motion of loop regions and the C-terminal peptide, as well as partial disruption of the second helix, is suggested to occur. Support for the existence of these motions can be found in amide exchange rate and spin relaxation time data.     

Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines.

A double-stranded RNA unwinding and modifying activity was found to be present in a wide range of tissues and cell types. The level of activity did not vary significantly with respect to the state of cell differentiation, cell cycle, or transformation. Thus, the unwinding and modifying activity, localized in the nucleus in somatic cells and capable of converting many adenosine residues to inosine, appears to be one of the housekeeping genes.     

Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex

1. Differential regulation, by dexamethasone, of glucocorticoid receptor gene expression was studied in three different neuronal cultures derived from hypothalamus amygdala, and cerebral cortex. 2. Cellular glucocorticoid receptor (GR) mRNA concentration was measured by hybridization using a 32P-labeled RNA probe complementary to a 2.2-kb fragment of the glucocorticoid receptor mRNA. Changes in the amount of GR mRNA were evaluated in relation to the content of beta-actin mRNA. 3. In cells derived from either hypothalamus or cerebral cortex, we observed a complex pattern of GR mRNA concentrations which were characterized by cyclic variations of GR mRNA content during continuous treatment with dexamethasone for up to 72 hr. 4. In contrast to cells derived from the hypothalamus where a persistent 30-40% reduction in GR mRNA levels was seen for up to a least 72 hr, we observed, in cells derived from the cerebral cortex, a sustained increased (1.4-fold) of the GR mRNA at this same time interval.     

Effects of platelet-activating factor on single potassium channel currents in guinea pig ventricular myocytes

Platelet-activating factor (PAF) has been implicated as one of the mediators of cardiac anaphylaxis. This phospholipid has been shown to have numerous effects on a variety of tissues, including the heart. Among these effects are alterations in the resting potential and generation of arrhythmias at very low concentrations. This suggests that PAF may modulate the activity of the background, inwardly-rectifying potassium current (I_K1). Thus, the effects of PAF on I_K1 were examined at the single channel level. Ventricular cells were isolated from adult guinea pig hearts and single channel currents recorded from cell-attached patches. PAF had substantial effects on the single channel currents at sub-nanomolar concentrations (10^–11 to 10^–10 M). PAF initially caused flickering of the channels, followed by a gradual prolonged depression of channel activity. Since these potassium channels play a major role in determining the resting potential and excitability of the cardiac cell, the effects of PAF on I_K1 may play a major role in the deleterious electrophysiological actions of PAF on the heart.Abbreviations I_K1 Inwardly-rectifying background potassium current - Lyso-PAF Lyso-platelet-activating factor - PAF Platelet-activating factor     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

The effect of salinity on the composition of fatty acid double-bond isomers andsn-1/sn-2 positional distribution in membrane phospholipids of a moderately halophilic eubacterium

We report the first study of the effect of NaCl on the double-bond isomeric composition of fatty acids and theirsn-1/sn-2 positional distribution in the membrane phospholipids of a moderately halophilic eubacterium. The major phospholipids, phosphatidylethanolamine and phosphatidylglycerol, ofVibrio costicola grown in 1M or 3M NaCl both have ansn-1 saturated,sn-2 unsaturated distribution of fatty acids. There is a greater effect of salinity on the fatty acid composition of phosphatidylglycerol compared with phosphatidylethanolamine. The fatty acids in phosphatidylethanolamine of cultures grown in 1M compared with 3M NaCl have the same unsaturation index and average chain length, but different double-bond isomeric compositions. In comparison, the fatty acid composition of phosphatidylglycerol is more unsaturated, with a different double-bond isomeric distribution, and has a shorter average chain length in cultures grown in 3M compared with 1M NaCl. The pattern of fatty acid isomers of 16:1 and 18:1 shows thatV. costicola uses the anaerobic pathway of fatty acid biosynthesis. The presence of the isomers 16:1c11 and 18:1c13 in the phospholipids of cultures grown in 3M but not in 1M NaCl indicates that external salinity affects the specificity of fatty acid synthetase in this moderately halophilic bacterium.     

Tissue architectural features for the grading of prostatic carcinoma

In research for the development of a computer-aided workstation for the objective grading of prostatic carcinoma, tissue architectural (histometric) features were analyzed in ten cases each of well-differentiated, moderately differentiated and poorly differentiated carcinoma (as subjectively graded by the consensus of a panel of experts). Sections were cut at 4 microns, stained by the Feulgen reaction and digitized by two different video-based photometric systems. Some images were interactively segmented, considering the histometric clues to be studied; others were automatically segmented by an expert system-guided technique. The latter procedure produced good results, with over 90% of the nuclei judged to be correctly segmented in 64% of the fields studied and over 80% in another 24% of the fields. While the number of nuclei per field provided some separation of well-differentiated from other lesions, the number of nuclei per gland distinguished between well-differentiated and moderately differentiated lesions. Simplicial decomposition of the images also provided a measure of the degree of differentiation, as did the "texture" of the nuclear placement, based on two run-length statistics. Combination of the run-length features distinguished the three categories of lesions with statistical significance. The results of this study provided insights into the problems (such as the effect of field boundaries) faced in the design of an computer-aided grading system. They also showed the value of expert system-guided scene segmentation and of such histometric features as the field cellularity and the number of nuclei per gland for the discrimination between lesions of different grades of differentiation.     

A physical map of the human salivary proline-rich protein gene cluster covers over 700 kbp of DNA

By using a linking library, we have experimentally linked, ordered, and spaced four of the six loci that constitute the human salivary proline-rich protein (PRP) multigene family. The methods used for mapping these four PRP genes may be useful in other multigene systems in which no probes unique to each member of genes are available, but in which some enzyme site that occurs only once in each member of the family can be found. The remaining two PRP loci have been provisionally mapped and linked within the gene cluster primarily on the basis of the resulting order giving a simple map. The order of the six loci that most simply accounts for our data is PRB2, PRB1, PRB4, PRH2, PRB3, and PRH1. The PRP gene cluster spans at least 700 kbp on chromosome 12 at p13.2. A scheme for the evolution of the cluster that requires an initial gene duplication followed by three unequal but homologous crossovers is given.     

Transforming growth factor-beta: multifunctional regulator of differentiation and development

Transforming growth factors-beta (TGF-beta) are 25 kilodalton (kDa) homodimeric peptides with multifunctional actions controlling the growth, differentiation and function of a broad range of target cells of both epithelial and mesenchymal derivation. They are expressed early in embryogenesis and their tissue-specific and developmentally dependent expression is strongly suggestive of an essential role in particular morphogenetic and histogenetic events. Five distinct TGF-beta s have been characterized so far, with 65-80% homology to each other. By using both molecular biological and immunohistochemical techniques, we are currently attempting to define specific sites of expression of the different TGF-beta s and to determine whether TGF-beta s 1-5 might have unique functions in development and in the mature organism. Comparative study of the promoter regions for the different TGF-beta s and for any particular TGF-beta in different species is also underway. Mechanistically, TGF-beta s act to control gene expression of their target cells, many of their actions converging on a complex, multifaceted scheme of control of matrix proteins and their interactions with cells; these effects on matrix are thought to mediate many of the effects of TGF-beta on development.     

Purification of multiple erythroid cell proteins that bind the promoter of the alpha-globin gene.

Three erythroid cell factors that bind the murine alpha-globin promoter were enriched more than 1,000-fold by conventional and DNA sequence affinity chromatography. Visualization of enriched polypeptides revealed simple patterns suggesting that each binding activity was purified. Two of the purified proteins, alpha-CP1 and alpha-CP2, have been shown previously to interact with distinct binding sites that overlap in the alpha-globin CCAAT box. Affinity purification of alpha-CP1 revealed seven polypeptides with Mrs raging from 27,000 to 38,000. In contrast, purified alpha-CP2 was made up of a polypeptide doublet with Mrs of 64,000 and 66,000. The third purified binding activity, alpha-IRP, interacted with sequences that formed an inverted repeat (IR) between the alpha-globin CCAAT and TATAA boxes. Affinity-purified alpha-IRP was made up of a single polypeptide with an Mr of 85,000. We confirmed that the purified polypeptides corresponded to alpha-CP1-, alpha-CP2-, and alpha-IRP-binding activities by UV cross-linking experiments (alpha-CP2 and alpha-IRP) or by renaturation of binding activity after elution of polypeptides from sodium dodecyl sulfate-polyacrylamide gels (alpha-CP1 and alpha-CP2). The apparent complexity of the polypeptides accounting for alpha-CP1 binding activity prompted a further physical characterization of this factor. Sedimentation of affinity-purified alpha-CP1 in glycerol gradients containing 100 mM KCl showed that all seven polypeptides migrated as a complex that cosedimented with alpha-CP1-binding activity. In contrast, when sedimented in glycerol gradients containing 500 mM KCl, alpha-CP1 dissociated into at least two components. Under these conditions, alpha-CP1-binding activity was reduced or lost. Activity was reconstituted, however, by combining fractions that were enriched in the two components. These results were confirmed by experiments in which we showed that alpha-CP1-binding activity can be recovered only by combining distinct sets of polypeptides that were isolated and renatured from sodium dodecyl sulfate-polyacrylamide gels. Our results suggest that the seven polypeptides visualized after affinity purification of alpha-CP1 interact to form a heterotypic complex (or set of complexes) required for alpha-CP1-binding activity.