Gene profiling study of G3139- and Bcl-2-targeting siRNAs identifies a unique G3139 molecular signature.

G3139 is a phosphorothioate oligodeoxyribonucleotide that is targeted to the initiation codon region of the Bcl-2 mRNA, which downregulates Bcl-2 protein and mRNA expression via an antisense mechanism. In previous work, we have demonstrated that the phenotype observed in several prostate and melanoma cell lines after treatment with G3139 appears to be Bcl-2 independent. In contrast, downregulation of Bcl-2 expression by a small interfering RNA (siRNA) produced little or no phenotype change. In the present work, we performed an Agilent oligonucleotide microarray assay on mRNA isolated from PC3 prostate cancer cells that were treated with two different oligonucleotide gene-silencing reagents. G3139 and a Bcl-2-targeting siRNA both downregulate Bcl-2 expression, but via different mechanisms. A side-by-side comparative analysis showed that the expression profile generated by these molecules differs substantially. The study revealed upregulation of the expression of stress-inducible genes in PC3 cells at 1 and 3 days after a 5-h transfection with G3139 complexed with Lipofectamine 2000. In contrast, only a very diminished stress response was seen 1 and 3 days after a 24-h transfection of siRNA/Lipofectamine 2000 complexes. These results highlight the profound differences in off-target effects in cells treated with the phosphorothioate oligonucleotide G3139 and with an siRNA targeted to the same gene, and provide further evidence that the mechanism of action of G3139 is not related to Bcl-2 silencing.

A microfluidic system for high-speed reproducible DNA sizing and quantitation.

Microfabrication technology was used to develop a system consisting of disposable glass chips containing etched channels, reagents including polymer matrix and size standards, computer-controlled instrumentation for performing electrophoretic separations and fluorescence detection of double-stranded DNA, and software for automated data analysis. System performance was validated for separation and quantitation reproducibility using samples varying in amount and size of DNA fragments, buffer composition, and salt concentrations. Several applications of the microfluidic system for DNA analysis have been demonstrated, such as of polymerase chain reaction (PCR) products, sizing of plasmid digests, and detection of point mutations by restriction fragment length polymorphism (RFLP) mapping.

The menstrual cycle and Raynaud's phenomenon.

Fluctuations in female sex hormones may be responsible for the high prevalence of Raynaud's phenomenon (RP) observed in premenopausal women. These hormones are known to act on central and peripheral thermoreceptors. In an attempt to establish whether cold sensitivity is altered during the menstrual cycle 50 premenopausal women were investigated. Of these, 26 had primary RP and 24 acted as controls. Each subject was exposed to environmental heating and cooling at three stages of the menstrual cycle to coincide with peaks and troughs in hormone levels. These stages were menstruation, periovulation, and during the midluteal phase. Finger hemodynamics was assessed by means of venous occlusion strain gauge plethysmography and fingertip temperature. Core temperature was assessed with an oral thermocouple. The results show that cold sensitivity was altered during the menstrual cycle in both groups with the fastest finger rewarming pattern during menstruation. Moreover, a significant difference was observed in core temperature between the two groups during the midluteal phase. As a group, subjects with RP failed to show a significant rise in core temperature following ovulation. The authors conclude that the menstrual cycle is associated with changes in the effect of cold on digital blood flow.

The effects of isolation on the mechanics of the human heel pad.

In previous studies on the mechanical properties of the human heel pad (Bennett & Ker, 1990; Aerts et al. 1995) the fat pad and part of the calcaneus was removed from amputated test specimens. The present study tested whether this procedure influences the mechanical behaviour of the sample. Intact amputated feet were therefore mounted on steel rods driven through the calcaneus and placed in a mechanical test situation (pendulum or servohydraulic material tester). The mechanical properties of the pad were determined for a series of experiments in which the pad was gradually freed from the foot in the way done by Bennett & Ker (1990) and Aerts et al. (1995). The results showed no observable differences in the mechanics of the pad by isolating it from the rest of the foot. Thus, in relation to human locomotion, the load-deformation relation of heel pads as described by Aerts et al. (1995) is the most appropriate to date.

The mechanical properties of the human heel pad: a paradox resolved.

In vivo and in vitro mechanical testing of the human heel pad gave apparently different properties for this structure: the in vivo stiffness is about six times lower, whereas the percentage of energy dissipation is about three times higher (up to 95% loss). It was postulated that this divergence must be ascribed to the lower leg being involved in in vivo heel pad testing. This hypothesis is presently evaluated by applying the two experimental procedures formerly used in the in vivo (an instrumented pendulum) and in vitro (an Instron servo-hydraulic testing machine) investigations on the same isolated heel pad samples. Instron load-deformation cycles mimicking pendulum impacts (i.e. 'first loop-half cycles') are first evaluated and then compared to real pendulum impacts. When performed properly, the pendulum test procedure reveals the same mechanics for isolated heel pads as the Instron does. The load-deformation loops are basically identical. Thus similar non-linear stiffnesses (about 900 kN m-1 at body weight) and comparable amounts of energy dissipation (46.5-65.5%) are found with both types of test, still being largely different from the former in vivo results (150 kN m-1 and 95%, respectively). Therefore, the present findings support the hypothesis that the presence of the entire lower leg in in vivo tests indeed influences the outcome of the measurements. It must be concluded that the previously published in vivo data, if interpreted for the heel pad alone, implied not only an incorrectly low resilience but also a value far too low for stiffness.

3'-Azidothymidine (zidovudine) inhibits glycosylation and dramatically alters glycosphingolipid synthesis in whole cells at clinically relevant concentrations.

Recent in vitro work with Golgi-enriched membranes showed that 3'-azidothymidine-5'-monophosphate (AZTMP), the primary intracellular metabolite of 3'-azidothymidine (AZT), is a potent inhibitor of glycosylation reactions (Hall et al. (1994) J. Biol. Chem. 269, 14355-14358) and predicted that AZT treatment of whole cells should cause similar inhibition. In this report, we verify this prediction by showing that treatment of K562 cells with AZT inhibits lipid and protein glycosylation. AZT treatment dramatically alters the pattern of glycosphingolipid biosynthesis, nearly abolishing ganglioside synthesis at clinically relevant concentrations (1-5 microM), and suppresses the incorporation of both sialic acid and galactose into proteins. Control experiments demonstrate that these changes do not result from nonspecific effects on either the secretory apparatus or protein synthesis. On the other hand, studies using isolated nuclei as a model system for chromosomal DNA replication show that AZTTP is a very weak inhibitor of DNA synthesis. These observations strongly suggest that the myelosuppressive effects of AZT in vivo are due to inhibition of protein and/or lipid glycosylation and not to effects on chromosomal DNA replication.

Acyclic guanosine analogs inhibit DNA polymerases alpha, delta, and epsilon with very different potencies and have unique mechanisms of action.

Acyclovir triphosphate, ganciclovir triphosphate and penciclovir triphosphate inhibited DNA polymerases alpha, delta, and epsilon. Each triphosphate preferentially inhibited pol delta, although ganciclovir triphosphate was the most impressive of the three; the Ki for inhibition of pol delta was 2 microM (competitive with dGTP), while the Kis for inhibition of pol alpha and epsilon were 80 and 140 microM, respectively. Each of the compounds was polymerized by pol alpha, delta, and epsilon. Incorporation of acyclovir triphosphate resulted in immediate chain termination, whereas incorporation of ganciclovir triphosphate often allowed polymerization of additional dNTPs. Interestingly, chain termination most often occurred after polymerization of just one additional dNTP onto the ganciclovir monophosphate. All three compounds were very weak inhibitors of DNA primase. Acyclovir triphosphate, however, was a unique inhibitor of the pol alpha-catalyzed elongation of primase-synthesized primers. Immediately after DNA primase synthesized a primer, pol alpha frequently incorporated acyclovir triphosphate with consequent chain termination. If, however, pol alpha did not immediately polymerize acyclovir triphosphate onto the primase-synthesized primer, further dNTPs were readily added and acyclovir triphosphate was incorporated much less frequently.

A thermographic study of paravertebral analgesia.

Six patients undergoing paravertebral blocks for chronically painful conditions of the chest wall were thermographically imaged so that the extent of cutaneous vasodilatation and hence sympathetic block could be correlated with the distribution of the somatic block. All blocks were performed by a single experienced operator, with a single percutaneous entry, using 15 ml of 0.5% bupivacaine at a mean level of T9-10 (range T7-8--T10-11), with radiological confirmation of correct needle placement. There was a mean distribution of the somatic block of five dermatomes (range 1-8), as evidenced by loss of pinprick sensation, with upper and lower limits of T6 and L3. The mean distribution of the sympathetic block was eight dermatomes (range 6-10), as evidenced by ipsilateral skin warming (p = 0.0005-0.001), with upper and lower limits of T5 and L3. No bilateral spread was observed. No significant postural changes in blood pressures were seen, although there was a small but significant decrease in supine heart rate (p = 0.05). This study demonstrates that a large unilateral somatic and sympathetic block is obtainable with a single thoracic percutaneous paravertebral injection. It challenges the suggestions that this method of analgesia is ineffective and hazardous, that a sympathetic component is a rare accompaniment and that the lumbar nerve roots are spared.

The cold hemiplegic arm.

BACKGROUND AND PURPOSE: Vasomotor changes occur in the arm after hemiplegic stroke. Previous studies have provided conflicting results, with most showing an increase in skin temperature of the hemiplegic arm. However, a number of patients complain of distressing coldness of the hemiplegic arm. METHODS: Eleven patients with symptomatic coldness and 10 patients with hemiplegia but no coldness were recruited. The severity of the symptom of coldness was compared by questionnaire with other common symptoms after stroke. A thermographic camera was used to record the finger skin temperature response to cold stress. Blood flow to both hands was also measured simultaneously by means of two plethysmographs. In all patients there were no symptoms in the unaffected arm, and this was used as a control. RESULTS: The symptom of coldness rated highly compared with other symptoms. In the symptomatic group the finger temperature on the hemiplegic side was lower at rest (median difference at rest, 0.65 degrees C; P < .0001) and at all times after cold stress. In the asymptomatic group the fingers on the hemiplegic side were colder at rest and after initial cooling (median temperature difference, 0.2 degrees C) but at no other time. Hand blood flow on the hemiplegic side was also decreased in the symptomatic group by 35%. This was not seen in the asymptomatic group. CONCLUSIONS: Coldness of the hand may be a severe and distressing symptom in some patients after hemiplegia. Symptomatic patients have lower finger skin temperatures at rest and after standard cold stress. These symptomatic patients also had reduced blood flow to the hemiplegic hand.

Calf thymus DNA polymerase alpha-primase: "communication" and primer-template movement between the two active sites.

The DNA polymerase alpha-primase complex replicates single-stranded DNA by first synthesizing a short RNA primer (primase) which is then further elongated by the incorporation of dNTPs (DNA polymerase alpha). While primase and pol alpha function independently prior to synthesis of an RNA primer, the two activities become coordinated after primer synthesis. After primase generates a primer-template, it moves from the primase active site to the pol alpha active site for further elongation without dissociating into solution. Intramolecular transfer occurs immediately after primer synthesis and is employed on both long templates such as poly(dT) and short synthetic templates (< or = 60 nucleotides). Primer-template transfer and elongation by pol alpha are rapid compared to primer synthesis. After pol alpha elongates the primer, primase reinitiates primer synthesis, and the cycle is repeated. However, if dNTPs are absent such that primer elongation cannot occur, further primase activity is inhibited after a single round of primer synthesis. This "negative regulation" of primase activity is mediated by the newly generated primer-template provided the following conditions are met: (1) Primase synthesizes the primer; (2) the primer is 7-10 nucleotides long and remains bound to the template; (3) the template is of sufficient length; (4) the primer-template dissociates slowly from the enzyme complex; and (5) the primer-template interacts with the pol alpha active site. Polymerization of multiple dNTPs by pol alpha rapidly reactivates primase; hence, negative regulation of primase activity likely ensures a new primer is not synthesized until the previous one has been elongated by pol alpha.

Inhibition of DNA primase and polymerase alpha by arabinofuranosylnucleoside triphosphates and related compounds.

Inhibition of DNA primase and polymerase alpha from calf thymus was examined. DNA primase requires a 3'-hydroxyl on the incoming NTP in order to polymerize it, while the 2'-hydroxyl is advantageous, but not essential. Amazingly, primase prefers to polymerize araATP rather than ATP by 4-fold (kcat/KM). However, after incorporation of an araNMP into the growing primer, further synthesis is abolished. The 2'- and 3'-hydroxyls of the incoming nucleotide appear relatively unimportant for nucleotide binding to primase. Polymerization of nucleoside triphosphates by DNA polymerase alpha onto a DNA primer was similarly analyzed. Removing the 3'-hydroxyl of the incoming triphosphate decreases the polymerization rate greater than 1000-fold (kcat/KM), while a 2'-hydroxyl in the ribo configuration abolishes polymerization. If the 2'-hydroxyl is in the ara configuration, there is almost no effect on polymerization. An araCMP or ddCMP at the 3'-terminus of a DNA primer slightly decreased DNA binding as well as binding of the next correct 2'-dNTP. Changing the primer from DNA to RNA dramatically and unpredictably altered the interactions of pol alpha with araNTPs and ddNTPs. Compared to the identical DNA primer, pol alpha discriminated 4-fold better against araCTP polymerization when the primer was RNA, but 85-fold worse against ddCTP polymerization. Additionally, pol alpha elongated RNA primers containing 3'-terminal araNMPs more efficiently than the identical DNA substrate.

Mechanism of DNA polymerase alpha inhibition by aphidicolin.

Synthetic oligonucleotides of defined sequence were used to examine the mechanism of calf thymus DNA polymerase alpha inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol alpha-DNA binary complex and thus should not be viewed as a dCTP analogue. Kinetic evidence shows that inhibition proceeds through the formation of a pol alpha.DNA.aphidicolin ternary complex, while DNase I protection experiments provide direct physical evidence. When deoxyguanosine is the next base to be replicated, Ki = 0.2 microM. In contrast, the Ki is 10-fold higher when the other dNMPs are at this position. Formation of a pol alpha.DNA.aphidicolin ternary complex did not inhibit the primase activity of the pol alpha.primase complex. Neither the rate of primer synthesis nor the size distribution of primers 2-10 nucleotides long was changed. Elongation of the primase-synthesized primers by pol alpha was inhibited both by ternary complex formation using exogenously added DNA and by aphidicolin alone.