Regions of bacteriophage T4 and RB69 RegA translational repressor proteins that determine RNA-binding specificity.

RegA protein of T4 and related bacteriophages is a highly conserved RNA-binding protein that represses the translation of many phage mRNAs that encode enzymes involved in DNA metabolism. RB69, a T4-related bacteriophage, has a unique regA gene, which we have cloned, sequenced, and expressed. The predicted amino acid sequence of RB69 RegA is 78% identical to that of T4 RegA. Plasmid-encoded RB69 RegA expressed in vivo represses the translation of T4 early mRNAs, including those of rIIA, rIIB, 44, 45, rpbA, and regA. Nucleotide sequences were determined for several T4 and RB69 regA mutations, and their corresponding repressor properties were characterized. All of the 10 missense mutations affect residues conserved between RB69 and T4 RegA. Two regions of RegA are especially sensitive to mutation: one between Val-15 and Ala-25 and another between Arg-70 and Ser-73. Sequence alignments and mutational data suggest that the region from Val-15 to Ala-25 is similar to helix-turn-helix domains of DNA-binding proteins and confers RNA-binding specificity upon RegA. The RegA691 protein (Ile-24----Thr) has an in vivo phenotype that appears to distinguish site-specific and cooperative binding modes of hierarchical RegA-mediated translational repression.     

Folliculostellate cells determine the susceptibility of lactotropes to estradiol's mitogenic action

Estradiol is known to increase lactotropic cell proliferation, but estradiol susceptibility varies among human populations and among various strains of rats. We had reported that folliculostellate (FS) cells regulate estradiol's mitogenic action on lactotropes; therefore, we studied their role in determining the susceptibility to estradiol in a high estradiol-responsive rat strain, Fischer 344 (F344), and in a low-responsive strain, Sprague Dawley (SD). Determination of total S-100-positive FS cells in the pituitary revealed that F344 rats have significantly more FS cells than do SD rats. Estradiol treatment did not change the number of FS cells in both F344 and SD rats. When cotransplanted with F344 pituitaries under the kidney capsule or cocultured with F344-derived lactotropes in vitro, FS cells derived from F344 rats increased estradiol's mitogenic action. They also increased estradiol's mitogenic action on SD-derived lactotropes in primary cultures. However, SD-derived FS cells failed to increase estrogen's action on F344- or SD-derived lactotropes. The levels of basic fibroblast growth factor production and secretion by TGF-beta 3 and estradiol were much higher in F344-derived FS cells than in SD-derived FS cells. However, the lactotropes' growth response to basic fibroblast growth factor was similar in both strains. These data suggest that cell-cell interaction between FS cells and lactotropes regulates estradiol's mitogenic action on lactotropes and also determines lactotrope susceptibility to the steroid.     

Contra-indications to metformin therapy are largely disregarded.

AIMS: To investigate the current metformin treatment practice and in particular to examine the consideration given to its contraindications. METHODS: A cross-sectional analysis of 308 consecutive Type 2 diabetic patients (mean age 66+/-11.3 years) previously treated with metformin on an outpatient basis and admitted to a German general hospital during the period from 1 January 1995 to 31 May 1998 because of acute disease or in order to optimize their diabetes management. All patients underwent a basic investigation comprising a documentation of their medical history, a physical examination, an electrocardiogram, and an extensive laboratory profile; 34% also had acute coronary angiography. RESULTS: On admission to hospital, 73% of the patients were found to have contra-indications, risk factors, or intercurrent illnesses necessitating discontinuation of metformin; 51% of these patients had several of these conditions. As major contra-indications to metformin, renal impairment was present in 19% of all patients, heart failure in 25%, respiratory insufficiency in 6.5%, and hepatic impairment in 1.3%. The risk factors to metformin included advanced coronary heart disease in 51%, atrial fibrillation in 9.8%, chronic alcohol abuse in 3.3%, advanced peripheral vascular disease in 2%, and pregnancy in 0.7%. As intercurrent illnesses, cerebral ischaemia occurred in 9.8% under metformin treatment and malignancies were diagnosed in 6.5%. The patients with contra-indications or requiring caution to metformin were significantly older and had previously been treated with more cardiovascular medication than those without such reservations (P<0.001). CONCLUSIONS: Despite the considerable risk of lactic acidosis in the majority of patients, no cases were observed.     

Structure of the DNA-binding region of lac repressor inferred from its homology with cro repressor.

It is shown that the amino acid sequence and the DNA gene sequence of the 25 amino-terminal residues of the lac repressor protein of Escherichia coli are homologous with the sequences of five DNA-binding proteins: the cro repressor proteins from phage lambda and phage 434, the cI and cII proteins from phage lambda, and the repressor protein from Salmonella phage P22. The region of homology between lac repressor and the other proteins coincides with the principal DNA-binding region of cro repressor. In particular, residues Tyr-17 through Gln-26 of lac repressor correspond to the alpha-helix Gln-27 through Ala-36 of cro repressor, which we have postulated to bind within the major groove of the DNA and to be primarily responsible for the recognition of the DNA operator region by the protein [Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, B. W. (1981) Nature (London) 290, 754--758]. By analogy with cro repressor, we propose that residues 17--26 of lac repressor are alpha-helical and that this helix and a twofold-related alpha-helix in an adjacent subunit bind within successive major grooves of the lac operator, which is in a right-handed Watson--Crick B-DNA conformation. Also, by analogy with cro repressor, we suggest that residues Thr-5 through Ala-13 of lac repressor form a second alpha-helix and contribute, in part, to DNA binding. The proposed structure for the DNA-binding region of lac repressor is consistent with chemical protection data and with genetic experiments identifying the probable locations of a number of the residues of the repressor protein that either do or do not participate in DNA binding.     

Short-course chemotherapy of tuberculosis with largely twice-weekly isoniazid-rifampin.

Although short-course, largely twice weekly chemotherapy for treatment of tuberculosis has been shown to be effective in other countries, when given under closely controlled conditions, it has not been adopted in this country where most patients are older and are treated as outpatients. Since January, 1976, 315 patients (mean age 55.5 years) with proven pulmonary tuberculosis have been treated with rifampin (RIF) 600 mg and isoniazid (INH) 300 mg daily for one month, followed by RIF 600 mg and INH 900 mg twice-weekly for another eight months, self-administered except for a few patients. By three months, 95 percent had converted to negative culture. There were only ten failures among 185 patients in whom final results could be assessed. There has been only one relapse during 1-21 months of follow-up in 175 patients. Serious side effects were few: six instances of jaundice, two of "flu-like syndrome," and one of thrombocytopenia. This form of initial therapy for tuberculosis is safe, effective, and economical.     

First-person action experience reveals sensitivity to action efficiency in prereaching infants

Do infants learn to interpret others’ actions through their own experience producing goal-directed action, or does some knowledge of others’ actions precede first-person experience? Several studies report that motor experience enhances action understanding, but the nature of this effect is not well understood. The present research investigates what is learned during early motoric production, and it tests whether knowledge of goal-directed actions, including an assumption that actors maximize efficiency given environmental constraints, exists before experience producing such actions. Three-month-old infants (who cannot yet effectively reach for and grasp objects) were given novel experience retrieving objects that rested on a surface with no barriers. They were then shown an actor reaching for an object over a barrier and tested for sensitivity to the efficiency of the action. These infants showed heightened attention when the agent reached inefficiently for a goal object; in contrast, infants who lacked successful reaching experience did not differentiate between direct and indirect reaches. Given that the infants could reach directly for objects during training and were given no opportunity to update their actions based on environmental constraints, the training experience itself is unlikely to have provided a basis for learning about action efficiency. We suggest that infants apply a general assumption of efficient action as soon as they have sufficient information (possibly derived from their own action experience) to identify an agent’s goal in a given instance.A hallmark of goal-directed action is its flexibility (1). Intelligent agents draw on causal and functional knowledge to update their action plans based on the constraints and affordances of the current environment. As a result, the same goal can be achieved with various means depending on the situation, and the same movements can reflect diverse goals. This flexibility poses a nontrivial inference problem for the outside observer seeking to uncover agents’ intentions and predict their future behavior.Humans nonetheless make sense of others’ behavior, and do so by recruiting a “theory of mind” that relates observable actions to subjective mental states (2). This intuitive theory relies on a key assumption that agents are rational, pursuing their goals with the most efficient means possible in a given context (3, 4). By assuming that agents pursue their goals efficiently, an observer can flexibly adapt expectations about others’ actions to the constraints of a given situation, and can interpret the same actions differently based on the context. This efficiency assumption thus constrains an otherwise underdetermined inference problem, providing a flexible schema for explaining and predicting action.     

Technique to determine contamination exposure routes and the economic efficiency of folded paper-towel dispensing

Handwashing and hand drying are key elements of infection control. Paper towels are generally accepted as the most hygienic means of drying hands and are often distributed from generic dispensers. Effective dispensing of towels is of importance economically and may influence infection control objectives if hands become contaminated during hand drying. In this study, a method to identify potential exposure routes for hand contamination and evaluate the efficiency of paper-towel dispensing is described and applied to 5 different folded paper towels using a generic wall-mounted dispenser. A total of 18 male and female participants of varying heights participated in pull testing of 400 paper towels each, in controlled hand-drying simulations. All events having the potential for hand contamination, including towel jamming, towels falling onto the floor, and incidental contact of paper exits, were monitored and documented. There was considerable variation in dispensing efficiency between different towel brands. One towel (Z) had significantly (P <.05) superior dispensing properties from the generic dispenser. Participants of a shorter height obtained a lower incidence of dispensing malfunctions using all towel products and type. The results indicated likely contamination exposure routes and wastage levels for each towel type. Environmental service managers and infection control practitioners should carefully consider, for economic and infection control reasons, the siting and design of towel dispensers and the types of towel purchased.     

Yeast genome-wide drug-induced haploinsufficiency screen to determine drug mode of action

Methods to systematically test drugs against all possible proteins in a cell are needed to identify the targets underlying their therapeutic action and unwanted effects. Here, we show that a genome-wide drug-induced haploinsufficiency screen by using yeast can reveal drug mode of action in yeast and can be used to predict drug mode of action in human cells. We demonstrate that dihydromotuporamine C, a compound in preclinical development that inhibits angiogenesis and metastasis by an unknown mechanism, targets sphingolipid metabolism. The systematic, unbiased and genome-wide nature of this technique makes it attractive as a general approach to identify cellular pathways affected by drugs.     

Antagonistic action of Bicoid and the repressor Capicua determines the spatial limits of Drosophila head gene expression domains

Bicoid (Bcd) is the anterior determinant in Drosophila. Accordingly, loss of Bcd causes loss of head and thorax and their replacement with posterior structures. bcd mRNA is maternally deposited at the anterior pole and Bcd forms an anterior-to-posterior (AP) concentration gradient. The expression of a series of zygotic head genes is thought to be differentially regulated by distinct threshold concentrations of the Bcd gradient. Thereby Bcd functions as a morphogen, instructing fields of cells to take on specific fates. Here, we show that spatial limits of anterior genes are also set in the absence of a Bcd gradient and depend on factors of the maternal terminal system. The receptor tyrosine kinase Torso (Tor), a key component of this system, is active in the pole regions of the embryo. Its activity downregulates the maternally deposited repressor Capicua (Cic), leaving high Cic activity in the central regions and decreasingly lower Cic activities toward the poles. We show that the positions of posterior boundaries of Bcd target genes are dependent not only on Bcd, but also on Tor-mediated Cic activity. The results indicate that Cic can mediate repression through distinct binding sites within a Bcd responsive enhancer and that gene activation by Bcd is antagonized by Cic. The activating and repressive effects of Bcd and Cic, respectively, are integrated by the Bcd target gene enhancer. We conclude that the spatial domains of head gene expression are determined by Bcd in concert with Tor-dependent repressors.     

Use of calcium ionophores to determine the effects of intracellular calcium on the action potential of canine cardiac Purkinje fibers.

The effects of calcium ionophores X-537A, A23187, and PR-47 on the action potential of canine cardiac Purkinje fibers were studied using standard microelectrode techniques. The ionophores hyperpolarize the membrane potential, eliminate the notch on the action potential plateau, decrease action potential duration, decrease plateau amplitude and duration, decrease spontaneous diastolic depolarization and excitability, and decrease the rate effect on action potential duration. These effects were shown to be independent of catecholamines, membrane-bound calcium, and the patency of the slow inward current channel. The effects of the ionophores depended on the presence of calcium in the external solution. A plausible interpretation of the data is that an ionophore-mediated increase in intracellular calcium shifts the voltage dependence of transmembrane potassium currents to more negative levels.     

Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA.

Several classes of trp repressor mutants were selected and analyzed in vivo. Mutants that produced repressors with either enhanced or reduced activity were obtained. One class of mutants produced inactive or slightly active repressors that were trans-dominant to the wild-type repressor. The amino acid substitutions in many of these repressors were clustered in a segment of the polypeptide that is homologous to the DNA recognition domain of the lambda cro repressor. A second functionally important region of the trp repressor was identified; this region could participate in L-tryptophan binding. Observations with trpR nonsense mutants suggest that the first 67 residues of the repressor polypeptide are sufficient for subunit association.     

Submillisecond folding of monomeric lambda repressor.

The folding kinetics of a truncated form of the N-terminal domain of phage lambda repressor [lambda 6-85] has been investigated by using the technique of dynamic NMR. lambda 6-85 has been shown previously to fold in a purely two-state fashion. This allows the determination of folding and unfolding rates from simulation of the exchange-broadened aromatic resonances of Tyr-22. The folding kinetics were determined over a range of 1.35 to 3.14 M urea. The urea dependence of both folding and unfolding rate constants is exponential, suggesting that the rate-determining step is invariant at the urea concentrations studied. The folding and unfolding rates extrapolated to 0 M urea at 37 degrees C are 3600 +/- 400 s-1 and 27 +/- 6 s-1, respectively. The observed lambda 6-85 folding rate constant exceeds that of other fast-folding globular proteins by a factor of 14-54. The urea dependence of the folding and unfolding rate constants suggests that the transition state of the rate-determining step is considerably more exposed to solvent than previously studied protein-folding transition states. The surprising rapidity of lambda 6-85 folding and unfolding may be the consequence of its all-helical secondary structure. These kinetic results clearly demonstrate that all of the fundamental events of protein folding can occur on the submillisecond time scale.     

Use of intracardiac recordings to determine the site of drug action in paroxysmal supraventricular tachycardia

Paroxysmal supraventricular tachycardia most often results from atrioventricular (AV) reentry using an accessory AV pathway (Wolff-Parkinson-White syndrome) or reentry within the region of the AV node. In AV reentry, using an accessory pathway, suppression of the tachycardia may be achieved by depressing either anterograde AV nodal conduction or retrograde accessory pathway conduction. Intracardiac recordings and programmed electrical stimulation have established that β-adrenergic antagonists and calcium channel blockers principally affect AV nodal conduction (anterograde limb of the reentrant circuit), whereas class IA and IC agents principally affect the accessory AV pathway (retrograde limb). Pharmacologic therapy has been more effective when directed at the limb in which conduction is most marginal at the tachycardia rate (weak limb). In individual patients, intracardiac recordings and programmed electrical stimulation can be used to identify the weak limb, indicating the class of agents most likely to be effective. Specialized techniques allowing direct recording of accessory pathway activation suggest that limitations in accessory pathway conduction may be explained by anatomic impediments. Conduction is most limited at the atrial interface of the accessory pathway in some patients, whereas in others the ventricular interface may be the limiting factor. Class IA and IC agents appear to have the greatest effect at sites where conduction is most tenuous, i.e., at the anatomic impediments.

Similar considerations apply to AV nodal reentry. Anterograde slow AV nodal pathway conduction is most often depressed by digitalis preparations, β-adrenergic antagonists, and calcium channel blockers, whereas retrograde fast AV nodal pathway conduction is more often depressed by class IA and IC agents. Intracardiac recordings and programmed electrical stimulation can also be used in these patients to identify the weak limb and direct pharmacologic therapy. Direct catheter recordings of AV nodal conduction remain elusive, limiting knowledge of the different conduction properties of the anterograde and retrograde limbs and the site(s) of drug action. Studies in progress, comparing the retrograde AV nodal conduction time during tachycardia with that during ventricular pacing at the same rate, suggest that the His bundle may be incorporated in the reentrant circuit in some patients. It appears that verapamil more readily depresses retrograde fast pathway conduction in these patients than in those in whom the His bundle does not form part of the reentrant circuit, but the reasons for this are unknown.     

Carvedilol's actions are largely mediated by endogenous nitric oxide.

Carvedilol's adrenergic antagonism does not fully explain its therapeutic actions. We therefore tested the hypothesis that its action is associated with an increase in NO synthesis. Wistar rats (male, 9 weeks, n = 10) anesthetized with sodium pentobarbital were used. Arterial NO concentration ([NO]), determined by chemiluminescence, and mean arterial pressure (MAP) were monitored throughout the experiment. In protocol 1), the effects of carvedilol (1 mg/kg, iv) were studied over a eriod of 90 min. In protocol 2), carvedilol was p administered, followed by the NO synthase (NOS) inhibitor L-NAME (5 mg/kg, iv) and by a second carvedilol administration. In protocol 1), carvedilol induced a significant fall in MAP (from 125,0 +/- 4,5 mmHg to 78.2 +/- 2.6 mmHg; p <0.001), reaching a minimum at t = 11.7 +/- 2.1 min and recovering 60 min afterwards (105.7 +/- 5.9 mmHg). Plasma [NO] varied in response to carvedilol in inverse proportion to MAP: baseline, 19.8 +/- 0.9 microM; t = 11.7 +/- 2.1 min, 32,3 +/- 2,3 microM; t = 60 min, 17.3 +/- 1.9 microM. In protocol 2), L-NAME administration blocked the effects of carvedilol (L-NAME: MAP, 129.9 +/- 5.0 mmHg; [NO], 13,1 +/- 2,3 microM. Post-L-NAME carvedilol administration resulted in MAP of 108.3 +/- 8.0 mmHg, NS, and [NO], 21.3 +/- 1.3 microM, NS. These results suggest that carvedilol increases plasma [NO], which is associated with a fall in MAP. Furthermore, carvedilol's hemodynamic action was blocked by NOS inhibition, suggesting that it depends on endogenous NO production, thus possibly explaining carvedilol's effects in hypertension and in cardiac failure.