Normal Template Research Articles

Tirofiban provides “platelet anesthesia” during cardiopulmonary bypass in baboons

Objective: Tirofiban (Aggrastat) is a reversible, nonpeptide inhibitor of platelet glycoprotein II/IIIa receptors. We tested the hypothesis that tirofiban preserves platelet number and function and shortens postoperative bleeding times in baboons after cardiopulmonary bypass. Methods: Four groups were studied: control, n = 12; low-dose tirofiban (0.1 μg/kg per minute), n = 7; high-dose tirofiban (0.3 μg/kg per minute), n = 7; and bolus tirofiban (15 μg/kg) followed by 0.1 μg/kg per minute during cardiopulmonary bypass, n = 7. After heparin, animals were perfused for 60 minutes at 50 ml/kg per minute and 37° C with a bubble oxygenator, roller pump, and peripheral cannulation. Hemodynamics, platelet count, platelet aggregation to adenosine diphosphate, and release of β-thromboglobulin were measured before tirofiban infusion, before heparin, after heparin before bypass, after 5 and 55 minutes of bypass, after protamine, and 60 minutes after protamine. Template bleeding times were measured at the same times except during cardiopulmonary bypass and 120 and 180 minutes after protamine administration. Platelet glycoprotein IIIa antigen was measured in Triton X-100 washes (Sigma Chemical Company) of the perfusion circuit after bypass. Results: High-dose tirofiban completely prevents platelet loss during cardiopulmonary bypass. β-Thromboglobulin release and sensitivity to adenosine diphosphate are significantly less than control at the end of bypass in all tirofiban groups. Template bleeding times return to preoperative values in both the low- and high-dose tirofiban groups 180 minutes after protamine administration and are significantly less than control bleeding times at both 120 and 180 minutes after protamine. Surface glycoprotein IIIa antigen does not significantly differ between groups. Conclusion: High-dose tirofiban completely preserves platelet number and improves platelet function during cardiopulmonary bypass in baboons and significantly accelerates restoration of normal template bleeding times after bypass(J Thorac Cardiovasc Surg 1997;113:182-93)

Identification and characterization of a hepatitis delta virus RNA transcriptional promoter.

Transcription and replication of hepatitis delta virus (HDV) RNA are performed by the cellular enzyme RNA polymerase II (Pol II). As DNA is the normal template for Pol II, the enzyme must undergo template switching. The mechanism for this is unknown, but since HDV RNA can form a rod-like molecule by extensive intramolecular base pairing, it has been suggested that a double-stranded region(s) of HDV RNA serves as a recognition site for Pol II. A bidirectional promoter has been identified previously on HDV cDNA (T. B. Macnaughton, M. R. Beard, M. Chao, E. J. Gowans, and M. M. C. Lai, Virology 196:629-636, 1993). In the present study, genomic RNA corresponding to this region was able to direct the synthesis of antigenomic RNA in a nuclear extract transcription reaction, whereas genomic RNA species containing a mutation that resulted in a replication-defective phenotype were unable to do so. Thus, this region, the location of which is defined as nucleotides 1608 to 1669 on the basis of a highly conserved structure, represents a RNA-RNA promoter. Computer analysis of the RNA secondary structure predicted that the promoter contains two bulge regions in a stem-loop structure which encompasses a GC-rich motif. This predicted model was confirmed by enzyme digestion and primer extension analysis. The promoter is located at one end of the rod and has some homology with the simian virus 40 late promoter. A number of other mutations were introduced within this region, and expression plasmids were constructed to examine the effects of mutations in the promoter on HDV replication. Disruption of the overall secondary structure, particularly the bulge regions, totally inhibited HDV RNA replication.

EPG treatment of sibilants in two Cantonese‐speaking children with cleft palate

ABSTRACT Children with disordered speech patterns have particular difficulties with fricatives and affricates, and it is also well documented that these segments are vulnerable in children with cleft palate (Grunwell, Sell & Harding, 1993). This is the case for both English‐speaking and Cantonese‐speaking children. The sibilant system in Cantonese differs from English in several ways: there are no voiced cognates, the affricates are alveolar, and there are only five sibilant segments in the system (/f/, /s/ /ts/ /tsh/ and /h/). We hypothesised that teaching the fricative (grooving) feature first, using electropalatography (EPG), would result in phonetic mastery of the affricate, since all of the features of the affricate would then be available to the child (alveolar/alveopalatal place, stopping plus grooving). The purpose of the study was to explore the efficacy of EPG as a treatment tool, to examine the rate and route of sibilant learning, and to examine the nature of affricates.The subjects were two Cantonese‐speaking children with repaired cleft palate, aged 5;8 and 7;8. Hearing was normal. The speech and language assessment included an oral motor examination, a story retell, an articulation test, and the Cantonese Electropalatography Protocol. The subjects were fitted with acrylic palates embedded with 62 silver electrodes, and habituated to them in situ until they were comfortable, prior to data collection. The assessment was audio‐ and video‐recorded and was completed within 1 1/2 hours.Perceptual analysis of whole‐word transcriptions were completed to construct the phonetic and phonemic inventories for each child. Electropalatographic data were analysed for the critical feature of frication (location and width of grooving) and those for affricates (place and amount of maximum constriction for the plosive, and features of grooving at the moment of release on to the fricative).Therapy included a combination of traditional and instrumental techniques. The children attended eight treatment sessions, each of 60 minutes' duration.Some cross‐linguistic differences were noted: these children maintained the feature of frication by bilabial fricatives. Both children made rapid progress from /s/ in isolation to /s/ in story retell and generalised the feature of frication to affricate. However, neither child achieved a normal template for [s]. The patterns for post‐treatment affricates showed that the location of the plosive component of the affricate was dependent on the location of the fricative, which was similar to the singleton [s].The rate and route of change seen in these children's speech patterns may further our understanding of the nature of affricates, their developmental route, and the difficulty they pose for children with speech disorders. These findings were used to explore five theories: motor theory (Fletcher, 1989); a coarticulation theory (Gibbons & Hardcastle, 1994); a frequency theory (Gibbon & Hardcastle, 1994); a matrix configuration theory (Manuel, 1990), and a sensory/feedback theory (Bzoch, 1979). The feedback theory and the matrix configuration theory were supported. The findings have implications for the description and treatment of speech disorders.

Oligonucleotide-Directed Switching of Dna Polymerases to a Dead-End Track

During DNA replication, the presence of oligonucleotides with partial homology to the template strand was shown to induce a switch of the polymerase from the normal template to the oligonucleotide. The latter acted as a dead-end template and led to abortive replication. The only prerequisite was that the oligonucleotide could form 7-9 base pairs with the newly synthesized DNA strand in order to switch templates. The switch occurred when base pairing of the oligonucleotide could take place with the 3'-end of the newly synthesized strand. These results show that oligonucleotides used in antisense or antigene strategies could have unexpected effects on replication. In addition, oligonucleotide-directed abortive replication might play an inhibitory role during PCR experiments on long DNA templates and lead to the amplification of truncated fragments.

Experimental fetal tracheal ligation and congenital diaphragmatic hernia: A pulmonary vascular morphometric analysis

The authors have previously shown that fetal tracheal ligation (TL) reverses the pulmonary hypoplasia in experimental diaphragmatic hernia (DH) by accelerating fetal alveolar growth. The purpose of this study was to determine if growth of the accompanying macroscopic and microscopic pulmonary vasculature is also accelerated. Eighteen fetal lambs were divided into three experimental groups: diaphragmatic hernia (DH), DH and simultaneous tracheal ligation ( DH TL ), and sham-operated controls (C). Animals were delivered near term, the lungs retrieved, and pulmonary capillary growth (5 to 50 μm in diameter) evaluated by standard morphometric techniques. Capillary ultrastructure was evaluated by electron microscopy. Nine additional fetal lambs of the same gestational age were equally divided into the same three groups and their lungs analyzed by pulmonary arteriography for evaluation of large vessel growth (<100-μm diameter). Computer digital analysis of angiogram lung slices showed that the total area of large vessels was increased in DH TL lungs when compared with DH lungs and decreased in DH lungs when compared with C lungs ( P = .003); however, the ratio of large vessel area per unit of lung area was similar in all groups. Microscopic morphometry of the capillary bed showed that the total number of capillaries was increased in DH TL lungs over both DH and C lungs ( P = .0001); however, the number of capillaries per alveolus ( cap alv ) was similar in all groups. In DH TL lungs, electron microscopy showed normal capillary wall structure and normal thickness of the capillary-alveolar interface, whereas in DH lungs, capillary structure was abnormal and the capillary-alveolar interface was thickened. DH animals had three times as many fully muscularized vessels as DH TL and twice as many fully muscularized vessels as C animals ( P = .0001). Furthermore, in DH lungs, 29% of vessels less than 100 μm in diameter were fully muscularized, whereas in DH TL and C lungs, there were no fully muscularized vessels less than 100 μm in diameter ( P = .0009). From these data the authors conclude the following: (1) Experimental fetal DH results in hypoplasia of both large pulmonary vessels and the capillary bed. (2) Fetal tracheal ligation is capable of reversing these effects by accelerating both large vessel and capillary growth based on total large vessel area and total capillary number. (3) With TL, large vessel growth remains proportional to overall lung growth based on normal values for large vessel area per unit of lung area, and capillary growth remains proportional to alveolar growth based on normal values for cap alv and cap/cm 2 lung tissue. (4) Experimental fetal DH results in increased muscularization of pulmonary arterial vessels as evidenced by an increased overall percentage of muscular arteries and by extension of muscle into normally nonmuscular vessels. (5) Fetal tracheal ligation reverses the increased muscularization of pulmonary arteries seen in experimental fetal DH. (6) By electron microscopy, capillary wall ultrastructure and the capillary-alveolar interface are abnormal in DH lungs and normal in DH TL lungs. The authors speculate that tracheal ligation exerts its effects by enhancing normal mechanisms of pulmonary development, thereby preserving the normal template for both pulmonary arterial and alveolar growth.

Nucleotide insertion and primer extension at abasic template sites in different sequence contexts.

Efficiencies of insertion and extension at a single site-directed abasic lesion, X, were measured while varying 5'- and 3'-template bases adjacent to X. The preference for insertion was found to be A > G > T approximately C, with the "upstream" (3'-neighboring) template base perturbing insertion efficiencies by an order of magnitude or more. Efficiencies of synthesis past the abasic lesion depended strongly on the "downstream" (5'-neighboring) template base and on the properties of the polymerase. HIV-1 RT favored "direct" extension of X.A > X.G > X.T > X.C, by addition of the next correct nucleotide. However, it was found that X.C, least favored for direct extension, was most favored for "misalignment" extension, occurring when the DNA structure in the vicinity of the lesion collapsed to realign a primer 3'-C terminus opposite a downstream template G site. Polymerase properties have an important role in copying abasic lesions. Drosophila DNA polymerase alpha, HIV-1, and AMV reverse transcriptases had "little" difficulty inserting opposite abasic lesions, with efficiencies comparable to misinsertions opposite normal template bases. However, AMV RT did not extent past the lesion using direct or misalignment mechanisms. Wild-type and mutant T4 DNA polymerases were used to show that although exonucleolytic proofreading inhibits lesion bypass, the presence of a highly active proofreading exonuclease is not sufficient to prevent bypass.

Platelet function defects associated with hemorrhage or thrombosis

Platelet dysfunction, especially acquired forms, is a common cause of hemorrhage, especially when associated with trauma or surgery. Although the hereditary platelet function defects are generally quite rare, hereditary storage pool disease is common enough to be suspected in an individual, usually a child, with characteristic historical and clinical findings. The acquired platelet function defects, especially those resulting from drugs, are common and should promptly be suspected in patients developing easy and spontaneous bruising, mild-to-moderate mucosal membrane hemorrhage, or unexplained bleeding associated with trauma or surgery. The template bleeding time is generally useful as a screening test of platelet function, but a normal template bleeding time, in the face of a suggestive history, suggestive clinical findings, or in a patient frankly bleeding, is not reliable, and platelet aggregation or lumiaggregation should be done in appropriate clinical situations. Also, prolongation of the template bleeding time is an unreliable predictor of clinical bleeding propensity. The mainstay of therapy for almost all these defects, if bleeding is significant, is the liberal infusion of appropriate numbers of platelet concentrates. The acquired platelet function defects should also be managed by attempts to treat or control the underlying disease, if possible, and offending drugs or potentially offending drugs should immediately be stopped.

Platelet function defects: a clinical review.

Platelet dysfunction, especially acquired forms, are common causes of hemorrhage, especially in association with trauma and surgery. Although the hereditary platelet function defects are generally quite rare, hereditary storage pool disease is common enough to be suspected in an individual, usually a child, with characteristic historical and clinical findings. The acquired platelet function defects, especially those resulting from drugs, are very common and should promptly be suspected in patients developing easy and spontaneous bruising, mild to moderate mucosal membrane hemorrhage, or unexplained bleeding associated with trauma or surgery. The template bleeding time is generally useful as a screening test of platelet function, but a normal template bleeding time, in the presence of a suggestive history, suggestive clinical findings, or in the patient frankly bleeding, is not reliable and platelet aggregation or lumi-aggregation should be done in appropriate clinical situations. The mainstay of therapy for essentially all these defects, if bleeding is significant, is the liberal infusion of appropriate numbers of platelet concentrates. The acquired platelet function defects, of course, should also be managed by attempts to treat or control the underlying disease, if possible, and offending drugs or potentially offending drugs should promptly be discontinued.

Recognition and Binding of Template-Primers Containing Defined Abasic Sites by Drosophila DNA Polymerase αHoloenzyme

Abstract Human DNA polymerase alpha holoenzyme follows an ordered sequential terreactant mechanism of substrate recognition and binding (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S.-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We confirmed this mechanism for the DNA polymerase alpha holoenzyme purified from Drosophila melanogaster embryos and studied the interaction of Drosophila pol alpha with synthetic oligonucleotide template-primers containing modified tetrahydrofuran moieties as model abasic lesions chemically engineered at a number of defined sites. Abasic lesions in the template had relatively little effect on the polymerase incorporation reaction at sites proximal to the lesion. However, incorporation opposite an abasic site was undetectable relative to that which occurred opposite a normal template nucleotide. Moreover, abasic residues in the primer region of the template-primer construct as far as 4 base pairs removed from the 3'-primer terminus prevented detectable nucleotide incorporation relative to that seen on an unmodified template-primer. Primer-region lesions had qualitatively similar effects whether they were located on the primer strand itself or on the complementary template strand. Data from polymerase incorporation experiments were corroborated by competitive binding assays performed under steady state reaction conditions. Results of these experiments suggested that polymerase binding to synthetic oligonucleotide template-primers was essentially unaffected by lesions located at sites that did not block incorporation. Lesions that did block incorporation apparently did so by abrogating template-primer binding. These observations have implications for understanding the mechanisms whereby DNA polymerase alpha recognizes noninformational template sites in vivo and prevents DNA synthesis from proceeding past these points.

Mechanism of chromium(VI) carcinogenesis

Since chromium(VI) is unreactive toward DNA under physiological conditions in vitro, the ability of carcinogenic chromium(VI) compounds to damage DNA depends on the presence of cellular redox components that reduce chromium(VI) to reactive species capable of interacting with DNA. We have examined the role of glutathione and hydrogen peroxide in chromium(VI)-induced DNA damage in vitro. Upon reaction with chromium(VI), glutathione produced chromium(V) and glutathione thiyl radical reactive intermediates, whereas hydrogen peroxide produced chromium(V) and hydroxyl radical. Reaction of DNA with chromium(VI) in the presence of glutathione resulted in binding of chromium and glutathione to DNA with little or no DNA strand breakage. Reaction of DNA with chromium(VI) in the presence of hydrogen peroxide produced the 8-hydroxydeoxyguanosine adduct and extensive DNA strand breakage in the absence of significant Cr-DNA adduct formation. These results suggest that the nature of chromium(VI)-induced DNA damage will be strongly dependent on reactive intermediates such as chromium(V), glutathione thiyl radical, and hydroxyl radical, produced by cellular components active in chromium(VI) metabolism. In order to assess the ability of chromium(VI)-induced DNA damage to affect the normal template function of DNA, we investigated the effects of chromium(VI) on steady-state mRNA levels of various genes in chick embryo liver in vivo, and compared the effects to the levels of DNA damage observed. Chromium(VI) induced DNA-protein and DNA interstrand cross-links in chick embryo liver in vivo and suppressed the induction of 5-aminolevulinic acid synthase and cytochrome P-450 mRNA expression by porphyrinogenic drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of nonlinear mathematical transformation of the normal electrocardiogram with the severity of coronary artery disease.

Signal processing was applied to normal ECGs to study whether the results could predict coronary artery disease (CAD) on angiography. ECGs determined to be normal by conventional criteria for 116 white males and 84 white females were subjected to signal processing. The technique applied nonlinear mathematical transformation to a segment of seven ECG leads which were photographically imaged, automatically digitized, normalized for time, and had topographical coordinate transformation using an IBM-XT microcomputer. The resulting curvilinear display on the computer monitor was termed a biopotential coordinate transformation (BCT). Successive superimposition of BCTs from normal ECGs of patients with normal angiograms developed boundaries of a normal template for each lead. Study BCTs were then compared to the normal templates to predict the presence or absence of CAD. Correlation of the BCT results with the severity of CAD for the males resulted in average sensitivities of 80%, 84.4%, and 91.7% for single, double, and triple-vessel CAD, respectively. Similarly, for the females average sensitivities were 59.1%, 73.9%, and 88.9%, respectively. In past studies on the same population, the BCT process detected CAD with average sensitivity of 85.1% and specificity of 81.8% for white males and average sensitivity of 73% and specificity 81% for white females. As such, computerized signal processing of ECGs may provide a noninvasive method for detection of CAD in patients with seemingly normal resting ECGs. The results of this study indicate that the sensitivity of the process increases in direct proportion to the severity of CAD. Further investigation is warranted for process refinement and verification.

Evaluation of brain-stem auditory evoked potentials using dynamic time warping

Dynamic time warping is a procedure whereby portions of a temporal sequence of values are stretched or shrunk to make it similar to another sequence. This procedure can be used to align the brain-stem auditory evoked potentials recorded from different subjects prior to averaging. The resultant warp-average more closely resembles the wave form of a typical subject than the conventional average. Dynamic time warping can also be used to compare one brain-stem auditory evoked potential to another. This comparison can show the differences that result from changes in a stimulus parameter such as intensity or repetition rate. When a patient's wave form is compared to a normal template, warping can identify the peaks in the patient's wave form that correspond most closely to the peaks in the normal template. Compared to an experienced human interpreter, warping is very accurate in identifying the waves of normal brain-stem auditory evoked potentials (error rate between 0 and 4%) and reasonably accurate in identifying the peaks in abnormal wave forms (error rate between 3 and 18%).

Bacteriophage T4 DNA primase-helicase. Characterization of the DNA synthesis primed by T4 61 protein in the absence of T4 41 protein.

The bacteriophage T4 61/41 protein primase-helicase is part of a seven T4 protein system needed for DNA synthesis in vitro. Although both 41 and 61 proteins are required for the synthesis and utilization of the normal pppApC(pN)3 pentanucleotide primer, we show in the accompanying paper (Hinton, D. M., and Nossal, N. G. (1987) J. Biol. Chem. 262, 10873-10878) that high concentrations of 61 protein alone carry out a limited, template-dependent oligonucleotide synthesis with the dimers pppApC and pppGpC as the major products labeled with [alpha-32P]CTP. At these high concentrations, 61 protein alone primes DNA synthesis by T4 DNA polymerase and the T4 genes 44/62 and 45 polymerase accessory proteins, or by Escherichia coli DNA polymerase I. The addition of T4 replication proteins other than 41 protein does not change the size distribution of oligonucleotides made by 61 protein. However, the primers used for DNA synthesis in the absence of 41 protein are not dimers, but rather trace quantities of longer oligonucleotides (5 to about 45 bases) which begin predominantly with pppGpC. These results show that 41 protein is required to prime with oligonucleotides beginning with pppApC and suggest that 41 protein, either alone or in conjunction with 61 protein, helps to stabilize the usual short pentamer primers on the template until they are elongated by the DNA polymerase. Moreover, since 61 protein by itself can only initiate DNA synthesis with primers beginning with pppGpC, but cannot make oligonucleotides starting with pppGpC on T4 DNA in which all the C is glucosylated and hydroxymethylated, both the T4 41 and 61 proteins are essential to prime DNA synthesis on their normal template. In our analysis of RNA-primed DNA, we demonstrate that although RNA primers at the 5' ends of DNA chains are relatively resistant to the 3' to 5' exonuclease of T4 DNA polymerase (Kurosawa, Y., and Okazaki, T. (1979) J. Mol. Biol. 135, 841-861), pppNpNpNpNpN oligomers are digested to a greater extent than the dephosphorylated pentamers NpNpNpNpN.

Minus-strand initiation by brome mosaic virus replicase within the 3′ tRNA-like structure of native and modified RNA templates

An RNA-dependent RNA polymerase (replicase) extract from brome mosaic virus-infected barley leaves has been shown to initiate synthesis of (−) sense RNA from (+) sense virion RNA. Initiation occurred de novo, as demonstrated by the incorporation of [γ- 32P]GTP into the product. Sequencing using cordycepin triphosphate to terminate (−) strands during their synthesis by the replicase generated sequence ladders that confirmed that copying was accurate, and that initiation occurred very close to the 3′ end. The precise site of initiation was further defined by testing the replicase template activity after stepwise removal of 3′-terminal nucleotides. Whereas removal of the terminal A did not decrease template activity, removal of the next nucleotide (C-2) did. Thus, initiation almost certainly occurs opposite the penultimate 3′-nucleotide (C-2) in vitro. The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3′-terminal A found on virion RNAs. The specific site of (−) strand initiation and normal template activity were retained for RNAs with as many as 15 to 30 A residues added to the 3′ end. However, only limited oligonucleotide 3′ extensions can be present on active templates. In order to assess the 5′ extent of sequences required for an active template, a 134-nucleotide-long fragment of brome mosaic virus RNA, corresponding to the tRNA-like structure, was generated. This RNA had high template activity, but a shorter 3′ (85-nucleotide) fragment was inactive. RNAs with various heterologous sequences 5′ to position 134 also showed high template activity. Thus, the 3′-terminal tRNA-like structure common to all four brome mosaic virus virion RNAs contains all of the signals required for initiation of replication, and sequences 5′ to it do not play a role in template selection.

Delayed DNA maturation, a possible cause of the elevated sister-chromatid exchange in Bloom's syndrome

Differences in behaviour between the 5-bromodeoxyuridine (BrdU)-substituted template strands in Bloom's syndrome (BS) and normal human fibroblasts have been investigated in order to elucidate the mechanism responsible for the elevated baseline sister-chromatid exchange (SCE) frequency in BS. Alkaline sucrose gradient analysis of the normal and BrdU-substituted DNA strands showed the former to be of higher mol. wt. and of mature size while the latter were of lower molecular size, resulting from breaks introduced during the repair of the BrdU with no differences discernible between BS and normal cells. The rates of removal of BrdU were similar in BS and normal cells, which indicates that the increased SCE level in BS is not due to different rates of repair of the BrdU. The maturation of newly synthesized DNA on a normal template is delayed in BS cells compared with normal cells although it is complete at 18 h, the time it is acting as a template for DNA synthesis. In the presence of a BrdU-substituted template the maturation although further delayed is complete in normal cells by 12 h but in BS cells is not complete even by 30 h, when the newly synthesized strand, due to cell cycle delay produced by the incorporation of BrdU, becomes a template in the next round of DNA synthesis. It is suggested that a similar delay in maturation probably occurs when a new strand containing BrdU is synthesized on a normal template in BS cells. When these strands act as a template they will contain two types of breaks--those due to BrdU repair and those due to delayed maturation. The latter will be responsible for the elevated SCEs in BS cells as the DNA replication forks move through them in a manner similar to that previously reported. The possible implications of differential delays in cell proliferation in BrdU, rates of BrdU removal and extent of DNA maturation in this syndrome are discussed.

Auditory brainstem responses (ABR) in multiple sclerosis.

Thirty-two multiple sclerosis (MS) patients, 10 males and 22 females, aged between 21 and 55, underwent pure-tone audiometry and testing of auditory brainstem response (ABR). Thirteen were classified as 'definite', 12 as 'probable' and 7 as 'possible', according to the McAlpine criteria. Each ear of each patient was tested monoaurally. The most common alterations were seen in the parameters of the cross-correlation between the normal template and the template of the individual MS patient. The second most altered parameters were those concerning the V wave which was absent in most cases but, when present, rarely showed latency-amplitude values falling outside the 90% confidence limit ellipses. Test-retest replicability and stimulations with increasing numbers of stimuli per second were useful in detecting intra-individual variability of waveform characteristics. When considering all the tests performed, 88% of the 'definite', 71% of 'probable' and 64% of 'possible' MS patients' ears showed ABR abnormalities. We stress the importance of a separate evaluation of the two ears due to the frequent unilateral alterations, and of an accurate evaluation of the ABR characteristics. We conclude that brainstem audiometry is a more reliable test for detecting demyelinating processes than is usually described in the literature.

Strand breaks arising from the repair of the 5-bromodeoxyuridine-substituted template and methyl methanesulphonate-induced lesions can explain the formation of sister chromatid exchanges.

5-Bromodeoxyuridine (BrdU)-induced sister chromatid exchanges (SCEs) are mainly determined during replication on a BrdU-substituted template. The BrdU, once incorporated, is rapidly excised as uracil (U), and the gap is repaired with the incorporation of BrdU from the medium, which leads to further repair. During the second S period in BrdU medium, this process continues as the strand acts as template. Experiments suggest that 3-aminobenzamide (3AB) delays the ligation of the gaps formed after U excision, resulting in enhanced SCE levels during the second cycle of BrdU incorporation. When normal templates of G1 cells are treated before BrdU introduction with methyl methanesulphonate (MMS), 3AB in the first cycle doubles the MMS-induced SCEs but has no effect on them during the second cycle. When the BrdU-substituted template is treated with MMS in G1 of the second cycle, 3AB again doubles the SCEs due to MMS and also enhances the SCEs resulting from delays in ligation of the gaps following U excision in the BrdU-substituted template. The repair processes of MMS lesions that are sensitive to 3AB and lead to SCEs take place rapidly, while the repair process of late repairing lesions that lead to SCEs appear to be insensitive to 3AB. A model for SCE induction is proposed involving a single-strand break or gap as the initial requirement for SCE initiation at the replicating fork. Subsequent events represent natural stages in the repair process of a lesion, ensuring replication without loss of genetic information.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro template activity of 0.3 mRNA from wild type and initiation mutants of bacteriophage T7.

Bacteriophage T7 0.3 mRNA synthesised and processed in vitro has been purified starting from the DNA of T7+ as well as from that of two initiation mutants of T7 (CR17 with a U----C transition in the initiation codon and CR35b whose potential Shine and Dalgarno (S-D) interaction is interrupted by a G----A transition). These mRNAs were used as templates to direct the binding of fMet-tRNA and the synthesis of 0.3 protein in both E. coli and wheat germ cell-free systems. The initiation codon mutant displayed approximately 50% inhibition of fMet-tRNA binding and 0.3 protein synthesis in both systems. The S-D sequence mutant, on the other hand, was found to be less affected than the initiation triplet mutant (20%-40% inhibition) in both fMet-tRNA binding and template activity in the E. coli system. In the wheat germ system, which does not make use of the S-D interaction, however, this mutant displayed normal template activity suggesting that the inhibition obtained in the E. coli system, albeit slight, is due to the impairment of the S-D interaction and not to an alteration of the mRNA secondary or tertiary structure caused by the base substitution.

DNA replication past pyrimidine dimers in the absence of repair

Post-UV DNA synthesis in Escherichia coli uvrA cells was studied. A low dose of UV radiation (0.07 J.m 2), which caused no degradation of the dimer-containing DNA, was used. This enabled us to make a direct comparison between DNA synthesis on the normal template and DNA synthesis on the UV-damaged template. There was no change in post-UV DNA synthesis kinetics during the first 60 min of post-irradiation incubation. A reduced rate of DNA synthesis was observed at later post-UV times when the dimers are expected to have passed through the normal replication complex. This reduced rate of DNA synthesis was associated with loos of the biological activity of the DNA. We suggest that the gaps opposite dimers rather than dimers per se interfere with normal replication, thus leading to cell death of uvrA recA bacteria.

Cell repair after liver injury: Stimulation of RNA synthesis, engaged polymerases, number of RNA transcribing molecules, and elongation rate in postischemic liver nuclei

Nuclei isolated from liver cells recovering from reversible (nonnecrogenic) ischemia show an increased RNA synthesis which is essentially sustained by the enhanced activity of the “engaged” (endogenous template-dependent) polymerases; “free” (exogenous templatedependent) polymerases do not change in activity. Chromatin isolated from postischemic nuclei, assayed with Escherichia coli polymerase, shows a normal template activity. Under experimental conditions known to maximize elongation, such as the presence of heparin and high ionic strength, the difference between normal and postischemic nuclei persists, suggesting that stimulation of RNA synthesis during the postischemic period may be caused in part by an increase in the number of polymerase molecules engaged in transcription. But under the same conditions stimulation of RNA synthesis decreases, implying that changes in elongation rate may also play a role in the postischemic effect. Measurements of [ 3H]UMP, derived from internal nucleotides, and [ 3H]uridine, derived from 3′-termini, and subsequent calculation of polynucleotide elongation rate and number of transcribing polymerase molecules confirm that postischemic recovery enhances RNA synthesis mainly by increasing the number of transcribing polymerases and in addition by stimulating the elongation rate of the α-amanitin-resistant polymerase I+III forms.