Actin in emerging neurites is recruited from a monomer pool.

Does actin in the emerging axons of regenerating neurons arise from the assembled or unassembled actin pool in the cell soma? We investigated this question by loading neurons with one of two fluorescently labeled molecules: rhodamine actin (r-actin) and rhodamine phalloidin (r-phalloidin). The assembly behavior of r-actin in vitro was identical to unlabeled actin. R-phalloidin binds tightly only to the filamentous form of actin (F-actin) and stabilizes filaments against disassembly. Hence, r-phalloidin-tagged filaments should be less likely to disassemble than r-actin-tagged filaments. Neurons of 10-d-old chick embryos were loaded with r-actin or r-phalloidin by triturating trypsinized dorsal root ganglia in isotonic sucrose containing the fluorescently tagged molecule. Isolated neurons were plated on glass coverslips in modified L15 medium containing nerve growth factor. Video images of the live cells on a thermoregulated stage were acquired with a computer imaging system. After 24 h in culture, the fluorescence distribution of r-phalloidin and r-actin was examined in live neurons of comparable morphology, neurite outgrowth, and intensity of somal fluorescence. Greater than 90% of the neurons labeled with r-actin (n = 81) contained detectable levels of fluorescence in emerging neurite fibers, often extending to the tip of the growing process. Less than 10% of the neurons labeled with r-phalloidin (n = 53) contained any fluorescence in the neurite fibers. In those that did contain fluorescence, the r-phalloidin usually was confined to the proximal segment of the neurite, and in no case was it found at the growing tip. Confocal microscopy and cooled CCD imaging of fixed neurons showed that all structures that incorporated r-actin or r-phalloidin also stained with bodipy phallacidin. This colocalization confirms the association of rhodamine-tagged species with F-actin. Our data support a model in which actin, needed in early stages of neurite outgrowth, arises from a pool in the soma that is capable of disassembly.


Neuronal protein NP185 is developmentally regulated, initially expressed during synaptogenesis, and localized in synaptic terminals.

Evidence is presented here that demonstrates the presence of NP185 (AP3) in neuronal cells, specifically within syn-aptic terminals of the central nervous system and in the peripheral nervous system, particularly in the neuro-muscular junction of adult chicken muscle. Biochemical results obtained in our laboratories indicate that NP185 is associated with brain synaptic vesicles, with clathrin-coated vesicles, and with the synaptosomal plasma membrane. Also, NP185 binds to tubulin and clathrin light chains and the binding is regulated by phosphorylation (Su et al., 1991). Based on these properties and the data reported here, we advance the postulate that NP185 fulfills multiple functions in synaptic terminals. One function is that of a plasma membrane docking or channel protein, another of a signaling molecule for brain vesicles to reach the synaptic terminal region, and a third is that of a recycling molecule by binding to protein components on the lipid bilayer of the synaptic plasma membrane during the process of endocytosis. In support of these premises, a thorough study of NP185 using the developing chick brain, adult mouse brain, and chicken straited muscle was begun by temporally and spatially mapping the expression and localization of NP185 in evolving and mature nerve endings. To achieve these objectives, monoclonal antibodies to NP185 were used for immunocytochemistry in tissue sections of chicken and mouse cerebella. The distribution of NP185 was compared with those of other cytoskeletal and cytoplasmic proteins of axons and synapses, namely synaptophysin, vimentin, neurofilament NF68, and the intermediate filaments of glial cells (GFAP). The data indicate that expression of NP185 temporally coincides with synaptogenesis, and that the distribution of this protein is specific for synaptic terminal buttons of the CNS and the PNS.


[Comparative measurements with breath alcohol measuring devices].

During a drinking experiment 12 persons were tested with two different breath-alcohol analysers. Both instruments fulfill essential requirements according to the expertise of the Federal Health Office concerning the forensic applicability of breath analysers. One instrument was equipped with different measuring systems (infrared system and fuel cell). The other instrument had only an infrared system and in each case two measurements were performed with this system. With both instruments breath temperature and breath volume were determined, so that the BrAC values could be compared after they were related to the reference temperature of 34 degrees C. Taking into consideration the course of BrAC as a function of time a measuring precision of all systems was found which can be characterized by a standard deviation of less than 0.01 mg/l.


[Glue solvent as the cause of a "breath alcohol value" of "1.96 promille"].

After sniffing of glue solvents (mixture of aliphatic hydrocarbons: boiling fraction 60-90 degrees C, ethylacetate and toluene) a blood alcohol concentration of 1.96 permille was indicated with an instrument using the infrared wavelength of 3.4 micrometer for the spectrophotometrical determination of breath alcohol. Testing of the individual solvents with a simple manual procedure showed linear relationships between concentrations in breathing air and the false positive breath alcohol values. Aliphatic hydrocarbons predominantly are responsible for the false positive breath alcohol determination. With 500 micrograms/liter a value of nearly 1.5 permille can be produced (using the factor of 2100 for the calculation of the blood alcohol concentration from breath alcohol). With ethylacetate or toluene up to 1 milligram/liter 0.5 permille ethanol in the blood will erroneously be found. The problem of recognition of crossreactive other gases by the measurement at two wavelengths (3.4 and 9.2 micrometer) is discussed.


[Modification of Alcomat breath alcohol determination by benzine].

We examined the period breath-alcohol values may be falsified owing to gasoline vapors with regard to the Alcomat currently used in routine tests. In a sober test person the breath-alcohol concentration was found to be 0.10/1000 24 min. after the inhalation of gasoline vapors, whereas the value measured after 28 min. was still 0.09/1000. 63 min. after the clothes had been moistened with gasoline, a breath-alcohol concentration of still 0.11/1000 could be identified immediately after inhalation.


[Criteria for the evaluation of programs for rehabilitation of apprehended drivers].

The preparation of criteria for the judgment of programs provided for rehabilitation (re-education) of delinquent drivers is in regard of the development of these measures--likewise in European foreign countries--necessary to secure their suitableness, their effectiveness, their controlled optimization, their credibility and their quality. Only programs (courses) can be suitable, which apply for the psychological knowledge about measures for behavior modification so that it relates perfectly to the target group. These measures have to be investigated for their effectiveness--especially if they result in legal consequences. Therefore the fundamental demand of Wittmann (1985) has to be repeated: Evaluation is a necessary, unquestioned task of all social institutions, which long for a rational manifestation and justification of their activity. Finally, behavior modification of delinquent drivers require a continuous safe-guarding of their quality within the scope of the acknowledged norms in Europe.


Purification and characterization of a 32-kDa protein that localizes to the sea urchin extraembryonic matrix, the hyaline layer.

We have purified a 32 kilodalton (kDa) protein that localized with isolated, intact hyaline layers prepared from 1-h-old embryos. The protein appeared not to bind calcium and was not quantitatively released from 1-h-old embryos in the absence of Ca2+ and Mg2+. Using polyclonal antiserum prepared against the 32-kDa protein, the antigen was detected throughout embryonic development. By the hatched blastula stage of development, the 32-kDa protein was replaced by a species of slightly smaller molecular mass. Quantitative determination indicated that the 32-kDa protein accounted for approximately 6% of the total protein present in the sea urchin egg. This result is suggestive of a structural role for the 32-kDa protein that is required throughout embryonic development, although perhaps in a modified form from the hatched blastula stage on.


Two squid skin proteoglycans each containing chondroitin sulfates with different sulfation patterns.

Two chondroitin sulfate containing proteoglycans, amounting to approximately 6% of the tissue proteoglycans, were isolated from the skin of the squid. They were almost completely extracted by 4 M guanidine hydrochloride in the presence of proteinase inhibitors, and then they were separated by DEAE-Sephacel chromatography and isolated by further chromatography on Sepharose CL-4B. Each proteoglycan contained two types of chondroitin sulfates that differed in their sulfation patterns. One proteoglycan (molecular mass (M(r)) 5.6 x 10(5)) contained, on the average, four chondroitins (M(r) 8.4 x 10(4)) and five chondroitin sulfates (M(r) 3.4 x 10(4)), whereas the other proteoglycan (M(r) 5.2 x 10(5)) contained three chondroitin sulfates (M(r) 1.1 x 10(5)) and five oversulfated chondroitin sulfates (M(r) 4.3 x 10(4)). The glycosaminoglycans were released from the proteoglycans by treatment with alkaline borohydride, separated from the oligosaccharides by chromatography on Bio-Gel P-30, and isolated by chromatography on DEAE-Sephacel and Sepharose CL-6B. Chondroitin sulfates were degraded by chondroitinase AC to an extent of 70% and consisted of significant amounts of disaccharides sulfated at C-4 of the galactosamine, disulfated disaccharides, and small amounts of nonsulfated disaccharides, as well as disaccharides that bore sulfates at C-6. Oversulfated chondroitin sulfate was degraded by chondroitinase AC to only 40% and contained appreciable amounts of disulfated and trisulfated disaccharides. The glycosaminoglycans also contained neutral monosaccharides; glucose was the predominant neutral sugar. A part of the oligosaccharides of both proteoglycans was of identical structure to that of chondroitin sulfate.


[Correlation of the lipophilicity of the active agents with their topical anti-inflammatory activity].

The present study concerns the relation between the therapeutic activity of some FANS in formulation for topical use and their lipophylic nature. The efficacy of topic formulations of three widely used FANS: cinnoxicam, piroxicam and diclofenac have been evaluated through the decrease of plantar edema induced by egg's white in the rat's hind paw. The volume increases after one hour were 55% in the group of rats treated with cinnoxicam, 64% in the animals treated with piroxicam and 79% in those treated with diclofenac, against 86% of control group. The partition coefficient (P) of the three active drugs have been detected. The P found for the three drugs 4.9, 3.3, 0.9 respectively demonstrate the direct correlation between the pharmacological activity of the topic formulation and its lipophylic nature.


In vivo behaviour of rat transferrin bearing a hybrid glycan and its interaction with macrophages.

Production of rat transferrin containing a single hybrid glycan was induced by treating rats with swainsonine, an inhibitor of alpha-mannosidase II. The principal component of this variant transferrin containing one sialic acid residue per mole of protein was separated from other forms of transferrin by anion-exchange chromatography, followed by lectin affinity chromatography. Transferrin bearing the hybrid glycan was degraded in vivo with a half-life of 14 h as compared with 40 h for transferrin containing a standard diantennary glycan. By using 125I-labelled tyramine-cellobiose, a label whose discharge from lysosomes is strongly retarded, organs rich in reticuloendothelial elements (liver, bone marrow, lungs, and spleen) were identified as the major sites of catabolism of the transferrin variant. The liver took up more 59Fe from the variant (26% of the dose in 90 min) than from control rat transferrin (12%). The excess iron uptake was reduced by the intravenous injection of either human transferrin or ovalbumin, and it was abolished by administering both. Macrophages from bone marrow and lungs degraded the transferrin variant in vitro. The degradation was significantly enhanced when transferrin receptors were blocked by human transferrin, and it was significantly reduced by ovalbumin and methyl glucopyranoside.


In vitro transfer of N,N'-diacetylchitobiose to glycoproteins in rat liver nuclei.

This work demonstrates that (N-acetyl[14C]glucosamine)2 is transferred from dolichyl pyrophosphate-(N-acetyl[14C]glucosamine)2 to endogenous nuclear glycoproteins. The (N-acetyl[14C]glucosamine)2 moiety is N-linked, since it can be released from the tryptic glycopeptides by N-glycosidase F and by hydrazinolysis, but not by beta-elimination. The biological significance of this direct transfer of N,N'-diacetylchitobiose to nuclear proteins remains to be elucidated.


Microbiological control of pharmaceutical products containing preservatives: drawbacks of the method of membrane filtration.

Microbiological assays carried out on membranes employed for the filtration of solutions of preservatives with concentration used in pharmaceuticals, demonstrated that such membranes, even after washing with 300 ml (100 ml X 3) of peptone water according to the guidelines of the Pharmacopoeia, maintain very frequently a remarkable antimicrobial activity revealed by inhibition zones and by disappearance or decrease of the number of colonies forming units. Such drawbacks can interfere negatively with the microbiological controls of pharmaceuticals containing preservatives.


Purpurogallin is a more powerful protector of kidney cells than Trolox and allopurinol.

Phase contrast and electron microscopic experiments demonstrated that oxyradicals generated with xanthine oxidase and hypoxanthine markedly damage rat kidney mesangial and porcine tubular epithelial cells. Purpurogallin, a phenol found in oak nutgalls, prolongs survival of the xanthine oxidase exposed renal cells three- to nine-fold longer than those without purpurogallin present. At levels equimolar to purpurogallin, either Trolox or allopurinol is less effective in delaying cell necrosis. Purpurogallin scavenges not only xanthine oxidase generated oxyradicals, but also non-enzymatically produced peroxyl radicals, more actively than equimolar levels of Trolox or allopurinol. Purpurogallin inhibits xanthine oxidase with severalfold higher potency than allopurinol and its more active metabolite oxypurinol. Therefore, purpurogallin is a stronger antioxidant than Trolox and a more potent inhibitor of xanthine oxidase than allopurinol as well as oxypurinol.


Slow troponin C is present in both muscle and nonmuscle cells.

A common primer was used for synthesis of cDNAs from both chicken fast and slow troponin C (TnC) mRNAs. Synthesis of double-stranded cDNAs and their amplification by polymerase chain reaction gave specific products corresponding to these mRNAs. This method was used for determining the presence of TnC mRNAs in various tissues. Our results show that while the fast TnC mRNA is expressed only in the muscle cells, slow TnC mRNA is expressed in a number of nonmuscle cells. Not all nonmuscle cells, however, express slow TnC mRNA. Liver and brain tissues showed the presence of high levels of this mRNA, while it was absent in chicken smooth muscle and embryonic skin. The slow TnC mRNA was very stable in cardiac muscle cells. It degrades with a half-life of approximately 94 h. The same mRNA was less stable in skeletal muscle and liver cells. The half-lives were found to be only between 13 and 16 h in these cells. Our results suggest that slow TnC mRNA may function as the nonmuscle isoform of this contractile protein. Since slow TnC mRNA is the only TnC isoform present in cardiac muscle, liver and brain, it is possible that besides its role in regulating contraction of striated muscle slow TnC can also function in processes other than muscle contraction.


Estrogen sulfotransferase distribution in tissues of mouse and guinea pig: steroidal inhibition of the guinea pig enzyme.

The highest total activity of estrogen sulfotransferase in guinea pig is in liver and the highest specific activities are in the adrenal and the midgestational chorion. Guinea pig gonads contain scarcely detectable activities. In CD-1 mice the highest specific activity is in testis and the highest total activity is in late placenta. Adrenals from both sexes and ovaries contain minimal activities, while liver and fetal membrane activities are remarkably low. In CD-1, DBA, C57BL, and BALB mice, qualitative patterns are similar. Purified or partially purified estrogen sulfotransferase from guinea pig adrenal and chorion were used to study the effect of a number of possible steroidal inhibitors. Considerable structural specificity is evident within a range of steroids, even among some which are not substrates. Pregnenolone is the most effective 21-carbon inhibitor and, in general, more highly hydroxylated forms are less inhibitory. Within a series of 21-, 19- and 18-carbon steroids, the structure of the A ring appears to be extremely important in regard to inhibitory effects.


Kinetics of inactivation of 4-aminobutyrate aminotransferase by 3-bromopyruvate.

3-Bromopyruvate inhibited 4-aminobutyrate aminotransferase (EC 2.6.1.19) from Pseudomonas fluorescens, apparently irreversibly. Kinetics of this inactivation were studied by continuously monitoring the enzyme reaction at 30 degrees C in the presence of inhibitor. Irrespective of how high an inhibitor concentration was present, a maximum rate of inactivation was eventually achieved (5.9 x 10(-3) s-1), indicating the formation of a reversible inhibitor-enzyme complex before the final inactivation step. The dissociation constant of this complex was found to be 6.5 microM. This affinity labelling by 3-bromopyruvate suggests the presence of essential sulphydryl groups on the enzyme, since this compound is known to preferentially alkylate cysteinyl residues.


Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus.

Virotoxins and phalloidin are peptides that induce actin polymerization in vitro. We have compared the effect of five virotoxins and phalloidin on the ultrastructure of spread preparations of Amoeba proteus cytoplasm. Like phalloidin, the five virotoxins induce polymerization of cytoplasmic actin. Moreover, the virotoxins, but not phalloidin, induce membrane fragmentation in small spherical vesicles. We, therefore, conclude that these virotoxins may have another membrane-bound target besides actin.


Amino acid pools in developing Chlamydomonas reinhardtii: vegetative cells, gametes, and mature zygotes.

Free amino acid pools were examined for cultures of vegetative cells, gametes, and mature zygotes of the unicellular green alga Chlamydomonas reinhardtii (Dangeard). The total pool of amino acids found in premature gametes of strains 137c+ (10.0 pmol-micrograms protein-1) and 137c- (10.8 pmol.micrograms protein-1) decreased to levels about half that seen in vegetative 137c- cells (19.8 pmol.micrograms protein-1). Following light activation, amino acid pools in these gametes increased to 18.7 pmol.micrograms protein-1 in 137c+ cells and 20.0 pmol.micrograms protein-1 in 137c- cells. With the exception of cystine, individual amino acid pools in these cells had increased once more to levels similar to those seen in vegetative cells grown in liquid medium. Levels of cystine remained one to two orders of magnitude lower than that seen in vegetative cells. Mature 137c+ and 137c- gametes mixed in solutions of either 2 mM cystine or 2 mM cysteine (half-cystine) suffered a 52-64% reduction, respectively, in the number of vis-à-vis conjugative pairs formed. This suggests that pools of endogenous cystine may play a role in the onset of mating. In zygotes levels of all amino acid pools, except histidine, were depressed; levels of cystine, valine, and phenylalanine were nondetectable in these cells.


Statistical analysis of trends in urban ozone air quality.

The purpose of this paper is to demonstrate the use of some statistical methods for examining trends in ambient ozone air quality downwind of major urban areas. To this end, daily maximum 1-hr ozone concentrations measured over New Jersey, metropolitan New York City and Connecticut for the period 1980 to 1989 were assembled and analyzed. This paper discusses the application of the bootstrap method, extreme value statistics and a nonparametric test for evaluating trends in urban ozone air quality. The results indicate that although there is an improvement in ozone air quality downwind of New York City, there has been little change in ozone levels upwind of New York City during this ten-year period.


Nonradioactive southwestern analysis using chemiluminescent detection.

Replacing radioactively labeled probes by nonradioactive ones and detection by chemiluminescence instead of colorimetry allows a nonhazardous handling and offers the possibility of easily reprobing filters in Southwestern analysis. Using the described procedure, we were able to determine the molecular weight of DNA-binding proteins and achieve a high signal-to-noise ratio.


A simple and sensitive enzyme-mediated assay of biotin.

Free biotin was quantitated by a competition by coating biotin-bovine serum albumin conjugate on a polystyrene microplate for binding to avidin-beta-galactosidase conjugate. The enzyme conjugate remaining on the plate surface as a result of the competition was detected by reaction with one of the following fluorogenic substrates, resorufin beta-D-galactoside and fluorescein di-beta-D-galactoside, in a fluorescence plate reader. Free biotin as little as 0.1 nmol can be routinely detected.


High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones.

Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to efforts to construct a contiguous cloned library of a genome. We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family. Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techniques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify. Similarly, several lambda bacteriophage clones constructed in the lambda DASH II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates. In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors.


Simultaneous synthesis of degenerate oligonucleotides of variable length.

Most mutagenic studies have emphasized the exchange of residues but disregarded the variation in length as the other aspect of protein variability. In this report a novel strategy to simultaneously synthesize degenerate mutagenic oligonucleotides of variable length is reported. Synthesis is done on a normal automated DNA synthesizer with modifications only in the program. Product of such synthesis can be used as a mutagenic oligonucleotide for construction of mutant proteins with variable length of inserts.


Taq DNA polymerase for labeling DNA using random primers.

The efficiency of labeling DNA with Taq DNA polymerase for probing nucleic acid blots was evaluated as an alternative to the more common procedure of using the Klenow fragment. The DNA was labeled with Taq DNA polymerase using random primers. The DNA was labeled specifically and efficiently. Synthesized DNA showed fragments of sizes smaller than those produced by the Klenow fragment and could be performed with as little as 0.5 pg of DNA. The use of Taq DNA polymerase appeared to be limited by the amount of radiolabeled nucleotide used and was more sensitive to non-optimized conditions in the reaction mixture than the Klenow fragment. The relative amounts of incorporated nucleotide in comparable conditions were, on occasions, 10% to 25% lower with Taq DNA polymerase than when using the Klenow fragment; nevertheless, the use of the Taq DNA polymerase to label DNA with random primers offers a very good alternative to the Klenow fragment as shown by this report.


Transient expression and histological localization of a gus chimeric gene after direct transfer to mature cowpea embryos.

Transient expression of a chimeric gus reporter gene was demonstrated in cowpea embryonic cells after direct gene transfer. This report shows that seed-derived embryos express the reporter gene at high frequency after passive or electroporation-mediated DNA transfer.


Circular dichroism user facility at the National Synchrotron Light Source: estimation of protein secondary structure.

The ultraviolet circular dichroism of a protein can be used to estimate the net fraction of its amino acids in different classes of secondary structure. Recent advances in the accuracy of such calculations have resulted from improved computational techniques, as well as extension of the spectral region analyzed to wavelengths less than 180 nm, a wavelength range beyond the limit of most laboratory-based circular dichroism spectrometers. We describe a spectrometer that uses UV radiation from the National Synchrotron Light Source at the Brookhaven National Laboratory to record circular dichroism spectra of proteins (and other biologically important molecules) in aqueous solution over the optimum wavelength range required for calculation of secondary structures. This instrument is available for use by scientists from academic, commercial and research institutions.


Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach.

Several improvements to the existing phosphorothioate-based site-directed mutagenesis methodology are reported, and here it is demonstrated that the new procedure is able to produce large deletions, insertions and point mutations rapidly and with very high efficiency. The time required for the polymerization step has been reduced by using T7 DNA polymerase to extend the mutant oligonucleotide primer-template. The reaction produces good yields of double-stranded closed-circular DNA and some partially polymerized template. The reaction was treated with T5 D15 exonuclease to selectively destroy partially polymerized single-stranded phage DNA that may otherwise contribute to an increased background of wild-type transformants. The use of these enzymes greatly facilitates the implementation of the phosphorothioate-based site-directed mutagenesis method by requiring less template DNA and by allowing all the in vitro manipulations to be completed in a day. In its present form the method may easily be automated, enabling large systematic site-directed mutagenesis projects to be undertaken.


Synthesis of cRNA probes from PCR-generated DNA.

We have compared RNA polymerase promoter activities in PCR-generated DNA fragments for use in the in vitro transcription of cRNA probes. Sense oligonucleotide primers, specific for the mouse acidic fibroblast growth factor gene, were synthesized with 5' extensions containing promoter sequences for the T7, T3 and SP6 RNA polymerase promoters. A common antisense primer was used with each of the promoter/aFGF primers to prepare PCR-generated DNA fragments (minigenes). In vitro transcription efficiency for each of these constructs was evaluated by incorporation of radioactivity into the cRNA products. We find that both the T7 and T3 promoters can direct the synthesis of cRNA probes of high specific activity from a PCR-generated DNA fragment, but that SP6 cannot. No detectable cRNA product was obtained using either T7 polymerase on the T3/minigene or T3 on the T7/minigene. Antisense cRNA probes, transcribed from minigene constructs were used for both Northern and in situ hybridization studies. A PCR-generated DNA fragment with RNA polymerase promoter sequences at each end provides a single template for synthesis in vitro of either sense or antisense cRNA probes.


An efficient and reliable method for cloning PCR-amplification products: a survey of point mutations in integrin cDNA.

A highly efficient, non-labor-intensive method for cloning DNA fragments produced by PCR amplification was used to carry out a rapid survey of potential point mutations in integrin alpha 6 cDNA from 17 different cell-type sources. The method includes glass powder purification of the PCR reaction mixture, followed by simultaneous treatment with T4 polynucleotide kinase and DNA polymerase I, and another glass powder purification. Sequences from multiple subclones of each cell type were readily generated, aligned and checked for mismatches. Several commonly used alternative procedures were compared for cloning efficiency and size-fidelity of inserted DNA fragments.


Minimal electrophoresis time for DNA sequencing.

This study presents a mathematical approach that allows one to determine the shortest electrophoresis time and migration path length required for DNA sequencing. The calculation was applied to the capillary electrophoresis of a DNA sequencing separation and showed that acceptable resolution could be obtained using a shorter path length than anticipated.


CircumVent thermal cycle sequencing and alternative manual and automated DNA sequencing protocols using the highly thermostable VentR (exo-) DNA polymerase.

CircumVent thermal cycle and standard DNA sequencing protocols utilizing the cloned and highly thermostable VentR (exo-) DNA polymerase are described. The thermal cycle sequencing procedures are advantageous because they allow fast and simple semiautomation of the sequencing reaction; make possible the direct DNA sequencing of PCR products, bacterial colonies and phage plaques; require only femtomoles of template DNA; eliminate the requirement of an independent primer annealing step; remove the requirement of denatured plasmids for sequencing double-stranded templates; and use a highly thermostable DNA polymerase for sequencing through potential recalcitrant secondary structure domains and large linear double-stranded DNA templates such as lambda derivatives. More standard methods of DNA sequencing (i.e., a one-step protocol and a labeling-termination protocol) are also presented. For each protocol, alternatives for choice of label and method of labeling are presented, including the use of 5' biotinylated primers for chemiluminescent DNA sequencing and fluorinated primers for automated sequencing using the BaseStation Automated DNA Sequencer.


A simple, efficient method to create a cDNA library.

The simplified "All In One Tube" protocol for constructing a cDNA library combines the advantages of the "Classic method" and the Okayama-Berg method while overcoming some of their drawbacks. In this method, adding adapters, linkers or enzyme digestion steps are no longer necessary after cDNA synthesis, thus making it quicker and especially useful when dealing with small samples of mRNA.


pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins.

We report on the construction of a plasmid, pGSTag, that directs the expression in E. coli of a glutathione S-transferase fusion protein that contains a high affinity phosphorylation site by protein kinase-A (PK-A). The fusion protein, following purification from crude bacterial lysates by substrate affinity chromatography, can be labeled in vitro to high specific activity with purified PK-A and 32P-gamma-ATP. Because labeling takes place while the fusion protein is immobilized on a solid support, the unincorporated label and enzyme can be washed away. Using the leucine-zipper domains of cAMP response element binding (CREB) proteins and CCAAT/enhancer binding protein (C/EBP)-like proteins as a model system, we show that the labeled protein, after elution from the affinity resin, can be used as a probe to detect interacting (dimerizing) species in a nitrocellulose-based ligand blot assay. The utility of this system for the creation of labeled protein probes is discussed.


Quantitation of radioactively labeled RNA by hybrid selection using biotinylated oligonucleotides.

We describe a procedure to quantify specific, radioactively labeled RNA sequences. This procedure combines hybrid selection of an RNA using biotinylated oligonucleotides with gel electrophoretic analysis of the selected RNA. We show that the hybrid selection procedure is specific and quantitative. It enriches a specific RNA sequence at least 600-fold. Specificity and sensitivity are increased to at least 10,000-fold enrichment by a combination of RNase T1 digestion of the RNA:oligonucleotide hybrid prior to selection, followed by gel electrophoretic fractionation of the selected RNA fragment. Furthermore, this modification allows one to quantify specific regions of an RNA transcript, as well as to monitor several different RNA sequences in one experiment. It is estimated that the sensitivity of this procedure is high enough to detect specific RNA sequences present at 1 part in 100,000.


Exonuclease cycling assay: an amplified assay for the detection of specific DNA sequences.

An assay is described in which an oligonucleotide probe is specifically digested by lambda exonuclease only when it is annealed to its complementary sequence. In this assay, a cycling effect occurs whereby a small amount of target sequence acts as a specific co-factor in the enzymatic degradation of a larger number of molecules of an oligonucleotide probe. This amplification principle is demonstrated and the effect of the oligonucleotide probe sequence investigated. The necessary steps needed to convert this effect into a useful diagnostic tool are discussed.


A more stringent choice of primers can improve the performance of fluorescent automated DNA sequencers.

Some primers frequently used in the double-stranded dideoxy DNA sequencing technique with radioactive markers are not suited for fluorescent detection. In fact oligonucleotides have a different annealing efficiency related to their base sequence, and this is reflected in nonequivalent results, particularly in fluorescent automated DNA sequencing. We present a method for the evaluation of primer performance in automated DNA sequencers and show its application to the search for a better set of primers for pBluescript vector.


Chemiluminescent detection of alkaline phosphatase in PhastGel.

A chemiluminescent assay has been applied to the detection of alkaline phosphatase on PhastGel containing lysates of preimplantation mouse embryos. The very sensitive detection capabilities reported for the chemiluminescent system led to the investigation of its applicability to the characterization of the alkaline phosphatases in one embryo or less and to compare the sensitivity of two different commercial alkaline phosphatase chemiluminescent assays to a colorimetric assay.


Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries.

We describe here a conceptually unique set of individual synthetic peptide combinatorial libraries (SPCLs), termed a positional scanning SPCL (PS-SPCL), that can be used for the rapid (i.e., a single day) identification of peptide sequences that bind with high affinity to antibodies, receptors or other acceptor molecules. The PS-SPCL described here is made up of six individual positional peptide libraries, each one consisting of hexamers with a single position defined and five positions as mixtures. As an example of the utility of such PS-SPCLs, the antigenic determinants recognized by two different monoclonal antibodies were correctly identified upon a single screening.


A simple computer program with statistical tests for the analysis of enzyme kinetics.

A simple computer program that calculates the kinetic parameters of enzyme reactions is described. Parameters are determined by nonlinear, least-squares regression using either Marquardt-Levenberg or Gauss-Newton algorithms to find the minimum sum of squares. Three types of enzyme reactions can be analyzed: single substrate reactions (Michaelis-Menten and sigmoidal kinetics), enzyme activation at a fixed substrate value or enzyme inhibition at a fixed substrate value. The user can monitor goodness of fit through nonparametric statistical tests (performed automatically by the computer) and through visual examination of the pattern of residuals. The program is unique in providing equations for activator and inhibition analysis as well as in enabling the user to fix some of the parameters before regression analysis. The simplicity of the program makes it extremely useful for quickly determining kinetic parameters during the data-gathering process.


A shell program for the design of PCR primers using genetics computer group (GCG) software (7.1) on VAX/VMS systems.

A number of software analysis packages for the design of PCR primers are available for PCs; however, software for users that depend on VAX/VMS operating systems is not available. By treating oligonucleotides as RNA molecules, I have designed an alternative means toward studying oligonucleotide interactions using software that is currently available from The Genetics Computer Group (GCG, Madison, WI). The oligonucleotide interactions with self and non-self are analyzed by the GCG FOLD program, a program which finds a secondary structure of minimum free energy for an RNA molecule. This approach allows the identification of self-priming primer pairs, and the interaction energies provide a guideline for the prediction of optimal PCR primers.


ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function.

This report describes the implementation of ProSearch, a computer program that can efficiently search for motifs in protein sequences. ProSearch currently uses motifs that are contained in the PROSITE database, but user-developed patterns can easily be added to any search. ProSearch can generate a report identifying the patterns present in a given protein sequence, their locations and, if desired, a short description of the identified patterns. The program is written in AWK (a small interpreted computer language), which can run on all computer platforms commonly found in laboratories. ProSearch can search a 348-amino acid protein for 690 patterns in less than 5 s on a typical workstation.


Application of the polymerase chain reaction to the ribonuclease protection assay.

We have developed a modified RNase protection assay in which the antisense RNA probe is prepared from a PCR-amplified DNA template rather than from a linearized plasmid DNA template. In this assay, an RNA polymerase promoter sequence is attached to the 5' end of the antisense PCR primer. Using this modified antisense primer in conjunction with the paired sense primer, PCR amplification generates a linear DNA template that includes an RNA polymerase promoter sequence. Transcription in vitro initiated by the incorporated promoter in the presence of RNA polymerase and ribonucleotide triphosphates produces a radiolabeled run-off antisense RNA transcript, which can then be used as probe for RNase protection analysis. Probes generated by this method obviate the need to subclone DNA sequences into transcription vectors for synthesis of antisense transcripts. Due to the simplicity of its design and the lack of need for subcloning, this strategy offers greater flexibility than conventional methods for the production of single-stranded RNA probes, and thus facilitates the implementation of the ribonuclease protection assay.


Practical applications in molecular biology of sensitive fluorescence detection by a laser-excited fluorescence image analyzer.

A new kind of fluorescence image analyzer was developed for a variety of uses, especially in molecular biology. Compounds labeled with fluorescent groups on a gel or nitrocellulose membrane are excited with 532 nm of light from a green laser. The fluorescence emitted passes through light-collecting fibers to a photomultiplier. Imaging data converted from the emitted light are analyzed by a microcomputer and stored on a magnetic optical disk. Dideoxy DNA sequencing was done with the same amount of DNA used for autoradiography, and the sequencing ladders obtained from gel scanning were automatically converted to sequence data by the analyzer. When an agarose gel was analyzed after electrophoresis, DNA stained with ethidium bromide was detected by the analyzer with higher sensitivity rather than by the conventional photographic method. Nylon and nitrocellulose membranes could be read by the analyzer, so blot hybridization experiments can be done without radioisotopes. High-quality computer storage of the imaging data from gel electrophoresis and hybridized membranes, including pulsed-field gels, make it possible to quantify image intensity and to construct many kinds of databases.


Progestins in breast cancer treatment. A review.

The two most widely used synthetic progestins in breast cancer treatment, medroxyprogesterone acetate (MPA) and megestrol acetate (MA), are reviewed with regard to pharmacological, endocrinological and clinical aspects. In high oral doses as second- or first-line endocrine therapy in advanced breast cancer, they give a similar response rate as tamoxifen (TAM) and aminoglutethimide (AG). The mechanism of action is probably complex. Considerable changes in serum levels of different hormones are induced by progestin treatment. The decrease of serum estrone sulfate (E1S) may be part of the therapeutic mechanism. Some studies suggest that the two drugs, MPA and MA, have a different mode of action, and possibly a low cross resistance. Randomized studies using the two progestins with a cross-over design may answer these questions. Further studies on the influence of progestin on different receptors and growth factors are warranted. To determine the most effective clinical dose of the two progestins, studies with increasing therapeutic doses are needed.


Statistical tests in medical research.

Various propositions have been made to improve the statistical quality of medical journals: using statistical referees, promoting better collaboration between statisticians and researchers, and teaching of basic statistics to clinicians. The most frequent errors found in medical articles are misinterpretation of p-values or non-significant results and confusion between statistical and clinical significance, inappropriate use of tests requiring precise assumptions, inappropriate or not controlled multiple testing and particularly testing of post hoc hypotheses, and overemphasis on p-values. Many errors arising from the misinterpretation of results of statistical hypothesis tests, the basic principles of this methodology are emphasized, and the usual fallacies found in the medical literature are reviewed.


Risk factors for screen-detected breast cancer. A case-control study.

Risk factors for breast cancer (BC) detected in mammography screening were sought using a questionnaire among 31,927 women aged from 40 to 74 years who attended screening. Data from 204 women with screen-detected BC were compared with those of 612 controls who did not have BC in screening. Mothers of women with BC had more often BC than those of the controls (8% vs. 3%, odds ratio, OR, 3.18, 95% CI 1.59-6.35). Women with screen-detected BC were older at their first childbirth (25.7 years vs. 24.7 years, OR 1.61, 1.14-2.33), and younger at menarche (13.6 years vs. 13.8 years, OR 1.38, 1.00-1.91), but there was no significant difference in the body mass index, number of pregnancies, breast size, smoking, or in the use of contraceptive pills between the cases and the controls. In multivariate logistic regression analyses, late age at the first childbirth, BC in the mother, and early age at menarche were independent risk factors for screen-detected BC but they appear to be of limited value in targeting screening in the female population aged from 40 to 74 in order to improve its cost-effectiveness.


Flow cytometry DNA analysis and prediction of loco-regional recurrences after mastectomy in breast cancer.

The study concerns whether DNA flow cytometry and estrogen receptor analysis might help predict which breast cancer patients, particularly node-positive ones, were at the greatest risk of developing loco-regional recurrence (LRR). Such patients would best benefit from postoperative radiotherapy following modified radical mastectomy and axillary lymph node dissection. After this type of surgery, 506 patients were followed up for a median time of nearly 5 years. Among the 235 patients given postoperative radiotherapy, the loco-regional control rate was 100% in N0 cases (n = 93), 94% in cases with 1-3 positive nodes (n = 90), 93% in cases with 4-9 positive nodes (n = 43), and 67% in cases with 10 or more positive nodes (n = 9). Among the 271 non-irradiated patients, the corresponding figures for loco-regional control were 91% in N0 cases (n = 141), 71% in cases with 1-3 positive nodes (n = 84), 65% in cases with 4-9 positive nodes (n = 31), and 67% in cases with 10 or more positive nodes (n = 15). Ploidy status, level of S-phase fraction, estrogen receptor content, and primary tumor size did not, in the present material, yield significant additional information with regard to the risk of LRR in the different nodal subgroups, a finding confirmed in multivariate analysis where the only significant predictor of LRR was the number of positive nodes (p = 0.01). Adjuvant tamoxifen treatment could not replace postoperative radiotherapy for achieving loco-regional tumor control, the overall rate of which was 81% among patients treated with tamoxifen only (n = 117), as compared with 98% among those also treated with radiotherapy (n = 54) (p = 0.003).


Breast cancer-associated antigen CA 15.3 in liver cirrhosis.

The tumor marker CA 15.3 was studied in 85 patients with liver cirrhosis. Nine patients (10.6%) had abnormal levels of CA 15.3 with the highest values in cases of Child's C patients. However, Child's classes were not significantly associated with the level of the antigen. We found significant correlations with some laboratory tests, especially IgA. All patients with an elevated CA 15.3 value also had abnormal levels of IgA, and multivariate analysis showed that IgA was the only independent factor associated with CA 15.3. Although IgA is a marker of alcoholic liver disease, other markers of alcoholism were not associated with CA 15.3. Cytolysis and cholestasis were not significantly associated with the CA 15.3 level, but liver dysfunction seemed to be involved. Liver disease does not substantially limit the usefulness of CA 15.3 in the cancer patient who also has liver cirrhosis, since both the percentage of abnormal values and the elevation of the serum levels are moderate in cirrhotic patients.


Development of resistance to antitumor chloroethylnitrosoureas in vitro in brain tumor cells.

Rat brain tumor cell lines (9L, C6-1, C6-2), human brain tumor cells (T98G), and HeLa S3 cells were studied to assess their acquired resistance to the chloroethylnitrosoureas (CENUs), 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and methyl-6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-glucopyr anosid e (MCNU), after 10 repeated exposures of a panel of different drug concentrations. Assay end-point was colony-forming ability after 24-h drug exposure. Intrinsic resistance was tested at the 10% survival dose (SD10) and C6-1, T98G, and HeLa S3 cell lines were 3 to 16 times more resistant to ACNU than 9L and C6-2 cell lines. After repeated exposures to ACNU, 9L and C6-2 cells acquired 2- and 5-fold resistance to ACNU respectively, whereas C6-1 and T98G cells retained a resistance almost equivalent to the respective parent cells. HeLa S3 cells also acquired resistance to ACNU, as evidenced by a 3.5-fold increase. The SD10 of the cells to MCNU ranged from 4.3 microM (C6-2 cells) to 151.7 microM (T98G cells). After long-term exposure to MCNU, all five cell lines became significantly resistant compared to their respective parent cells. The easily obtained acquired resistance to CENUs suggests a clinical disadvantage of continual and repeated adjuvant monochemotherapy with these agents.


The effect of ondansetron on radiation-induced emesis and diarrhoea.

Fractionated radiotherapy of malignancies in the abdomen induces nausea and vomiting in approximately 50% of the patients. During abdominal irradiation the damaged gastrointestinal mucosa releases 5-HT with ensuing activation of 5-HT3 receptors which may explain the nausea and vomiting. Ondansetron is a new 5-HT3-antagonist with antiemetic properties. In this consecutive study, 33 patients receiving fractionated upper abdominal irradiation (> or = 100 cm2, 1,8-4 Gy daily dose for a mean of 13 days) were treated with ondansetron (8 mg t.d.s. p.o.). Emesis was completely controlled in 26/33 (79%) patients throughout their radiation course, which embraced 628 (94%) treatment days. Ondansetron was well tolerated. Eleven patients developed mild constipation. No patients experienced diarrhoea (a common distressing side-effect of abdominal irradiation). It is suggested that ondansetron can be of value in preventing emesis in patients receiving fractionated radiotherapy. The possible beneficial effect in preventing diarrhoea must be further evaluated.


A PET system based on 2-18FDG production with a low energy electrostatic proton accelerator and a dual headed PET scanner.

We have developed a comparatively inexpensive PET system, based on a rotating scanner with two scintillation camera heads, and a nearby low energy electrostatic proton accelerator for production of short-lived radionuclides. Using a 6 MeV proton beam of 5 microA, and by optimization of the target geometry for the 18O(p,n)18F reaction, 750 MBq of 2-18FDG can be obtained. The PET scanner shows a spatial resolution of 6 mm (FWHM) and a sensitivity of 80 s-1kBq-1ml-1 (3 kcps/microCi/ml). Various corrections are included in the imaging process, to compensate for spatial and temporal response variations in the detector system. Both filtered backprojection and iterative reconstruction methods are employed. Clinical studies have been performed with acquisition times of 30-40 min. The system will be used for clinical experimental research with short- as well as long-lived positron emitters. Also the possibility of true 3D reconstruction is under evaluation.


The objective response of brain metastases on radiotherapy. A prospective study using computer tomography.

A prospective study was performed to establish the objective response of brain metastases after radiotherapy. Twenty-five patients with a total of 53 metastatic lesions were evaluated approximately 6 weeks after completing the treatment (30 Gy/2,5 weeks). Clinical response was seen more often than objective response, due probably to concomitant use of corticosteroids and to most patients having multiple metastases. Clinical response, although important for the patient, does not well reflect the objective response. In 48% of the cases there was agreement between the clinical and the radiodiagnostic findings. The tumor size was an important prognostic factor for prediction of complete response. The majority of patients who achieved complete remission of at least one of the metastatic lesions, did not show recurrence of the neurological symptoms.


Tolerance of rat small intestine to localized single dose and fractionated irradiation.

The tolerance of rat small intestine to localized single-dose and fractionated irradiation was assessed. In 168 rats, bilateral orchiectomy was performed and a loop of small intestine was transposed to the left part of the scrotum. Beginning 3 weeks postoperatively, single dose (18-24 Gy) or fractionated (4.2 Gy or 5.6 Gy per fraction) x-irradiation was delivered to the transposed intestine. The animals were observed for complications, and groups of animals were killed 2 and 26 weeks after completion of irradiation for assessment of injury. Mortality (i.e. the occurrence of lethal intestinal complications) and a semiquantitative histopathologic scoring system were used as endpoints to assess the degree of radiation injury. The most frequent intestinal complications were enterocutaneous fistula formation and intestinal obstruction. Logistic regression analysis ov complications data was used to estimate LD50 values and the alpha/beta ratio. There was good correlation between histopathologic scores and the incidence of lethal complications. The estimated LD50 values were 22.1 +/- 0.5 Gy, 37.0 +/- 4.4 Gy and 51.0 +/- 5.3 Gy for the single dose regimen and the fractionated regimens of 5.6 Gy and 4.2 Gy respectively. The estimated alpha/beta ratio was 10.7 +/- 2.4 Gy. The goodness of fit of the linear-quadratic isoeffect model to our data was satisfactory. Our results indicate that acute mucosal damage may be pathogenetically involved in the development of intestinal complications.


[The SAVE trial: rational basis, results, and reflections].

Recent concepts about post-infarction left ventricular remodeling, which is the basis for heart failure in these patients, as well as its prevention by ACE inhibitors are briefly summarized. Those data were the rationale for the SAVE trial. The most important initial aspects of this trial (general objective, pre-specified endpoints, inclusion and exclusion criteria, etc.) are then described as well as the basal characteristics of the respective cohort. The most important results of the SAVE trial, now in press, are subsequently presented. Several clinical guidelines, derived from these results, are then suggested. Finally, some new questions, both clinical and pathophysiological, and originated by the results from the SAVE results, are commented.


[Variability of heart rate: a perspective].

The present work is an attempt to provide a general overview on the current methods and applications of heart rate variability (HRV), starting from the underlying mechanisms of cardiac rhythmic automaticity and the influence on heart rate of biological modulators, namely autonomous nervous system. Different evaluation methods for HRV are compared in what concerns the duration of the records under analysis and the mathematical approach (spectral/non-spectral). Finally, clinical applications of HRV are mentioned, some of them the Department is particularly interested in, namely coronary heart disease, heart failure and diabetic neuropathy. The authors conclude, minding some aspects susceptible of discussion in the future of this technique.


[Pericarditis in the acute phase of myocardial infarction: incidence and clinical significance].

To evaluate the incidence and the clinical significance of pericarditis in the acute myocardial infarction. Retrospective study. The Coronary Care Unit of a University Hospital. We have studied 668 consecutive patients with their first acute myocardial infarction admitted at the Coronary Care Unit, Hospital General de Galicia, Santiago de Compostela, Spain, in the years 1983 to 1988. Pericarditis was defined as the presence of a pericardial friction rub on auscultation during the hospital course. Pericarditis was noted in 86 patients (12.8%), who were considered as group A. The remain 582 patients were considered as group B. Statistical analysis was carried out using the BMDP statistical package. Pericarditis occurred in 12.8% of the patients. Patients with, compared to those without, pericarditis had a lower age (59.0 +/- 12.4 years; p = .0005), and a higher percentage of males (86.1% versus 75.6%; p = .038), an a higher percentage of smokers (63.9% versus 48.6%; p = .01). The delay to the hospital admission was greater in group A (12.6 +/- 18.5 hours versus 8.0 +/- 11.7 hours; p = .0024). Pericarditis more often occurred in the setting of anterior wall myocardial infarction and in Q-wave infarct. The group A had a higher CPK peak (1877.5 +/- 1548.9 UI/L versus 1240.2 +/- 961.5 UI/I; p = .001) and a higher peak of CK-MB (213.7 +/- 134.7 UI/L versus 160.8 +/- 112.9 UI/L; p = .001). In-hospital mortality was significantly lower in group A (6.9% versus 17.2%; p = .016). The multivariate analysis by stepwise logistic regression identified the Q- wave myocardial infarct, the age, the delay to the hospital admission, the peak of MB creatine kinase and location of infarct as the only independent predictive variables for the pericarditis occurrence. We conclude that the pericarditis in the setting of Q-wave myocardial infarction, with anterior wall location, and is related to transmural extension of the myocardial necrosis.


[Combined infarction: angiography and electrocardiographic diagnosis].

Combined infarction can be defined as the presence of ST segment elevation simultaneously in anterior and inferior leads. Their possible anatomical and physiopathological causes are suggested. Four cases of combined infarction, three of them in previously asymptomatic patients are presented, with their electrocardiographic and angiocoronarography patterns. Acute phase and 24 hours ECG ST changes are analysed and the possibility of an indirect approach to the angiographic diagnosis, is discussed.


[Pulmonary embolism--mortality risk].

We conducted a retrospective analysis on 311 patients with clinical diagnosis of pulmonary embolism (PE) in a period of 3 years. 163 patients were excluded based on clinical-laboratorial criteria. The remaining 146 patients had a median age of 69 years (range: 30-91 years). 54% of the patients were male. We found dyspnea (94%), abnormal cardiopulmonary observation (89%), risk factors for venous thromboembolism (74%), tachycardia (53%), cyanosis (49%), and neck vein distension (45%) to be the most frequent findings. 64% of the patients had heart failure, 32% had myocardial ischemia, 13% had cancer, and 11% had myocardial infarction. Lactic dehydrogenase (LDH) was higher than two-fold in 54% of the patients. There was severe hypoxemia in 55% of the cases and hypocapnia in 43% of the cases. Creatinine phosphokinase (CPK) was elevated in 16% of the cases. Electrocardiography was suggestive of PE in 37% of the cases. Echocardiography showed right heart dysfunction in 30% of the cases, 92% of the patients were treated with heparin, 37 patients (25%) died, 54% of which during the first 4 days after admittance. Trying to define an index of mortality in PE we evaluated all patients by discriminant analysis coming up with 14 items with good discriminative power. By approximation of their odds-ratios we determined how many points would correspond to each item in the total sum.(ABSTRACT TRUNCATED AT 250 WORDS)


[Transesophageal echocardiography study of mitral valve prosthesis].

Transesophageal (TEE) and Transthoracic (TTE) comparative echocardiographic analysis of patients with mitral valve prosthesis. Prospective study. In hospital and out patients with mitral prosthesis in a follow-up study by the Cardiology Department and referred to the echocardiographic laboratory of Gregorio Marañon General Hospital, Madrid. We studied 90 consecutive patients with mitral prosthesis diagnosis. Comparative and prospective echocardiographic study of transthoracic and transesophageal techniques in mitral prosthesis pathology. TTE diagnosed 18% of dysfunctioning mitral prosthesis patients compared to 56% of cases identified by TEE. TTE observed 1% of mitral prosthesis vegetation compared to 8.8% by TEE. TEE diagnosed left atrial thrombosis in 2.2% and TEE in 10%. Left atrial spontaneous dynamic contrast was identified exclusively by TEE in 55% of all cases. Paravalvular mitral prosthesis leak was correctly identified exclusively by TEE in 20% of cases. TEE has a greater diagnostic capacity compared to TTE in mitral prosthesis patients. This technique can give a greater security in evaluating mitral prosthesis thrombi, vegetations and leak, establishing a more precise diagnosis of mitral prosthesis dysfunction. The authors concluded that TEE is the technique of choice in noninvasive evaluation of patients admitted with the suspicion of mitral prosthesis dysfunction.


[Hemorrheologic changes in the hypertensive elderly patient].

The aim of this work is to make a review of the papers about hemorheology and hypertension on the aged. The paucity of the works did not allow any conclusions. However, some of the drugs frequently used have tight links with hemorheology, which must be in our minds when dealing with old people.


Nonpharmacological treatment of supraventricular tachycardia.

Although pharmacological treatment is often effective in preventing or controlling attacks of tachycardia it is palliative rather than a cure. Some Arrhythmias are refractory to drug therapy and long-term antiarrhythmic agents are associated with side-effects, life-threatening toxicity, poor patient compliance and considerable cost. On the other hand, enhanced knowledge of electrophysiological mechanisms and advanced technology have resulted in the more and more widespread use of several nonpharmacological methods of treating arrhythmias. It is the purpose of this review to discuss the present status of nonpharmacological therapy of supraventricular tachycardia and comment upon its current role in the management of patients presenting with these arrhythmias.


Biological complexes of poly-beta-hydroxybutyrate.

Short-chain complexed poly-beta-hydroxybutyrate, 130-170 monomer units, is a ubiquitous constituent of cells, wherein it is usually associated with other macromolecules by multiple coordinate bonds, or by hydrogen bonding and hydrophobic interactions. This conserved PHB has been isolated from the plasma membranes of bacteria, from a variety of plant tissues, and from the plasma membranes, mitochondria, and microsomes of animal cells. In bacterial membranes, PHB has been found complexed to the calcium salts of inorganic polyphosphates, and to single-stranded DNAs. The ability of PHB to solvate salts, consisting of cations having high solvation energies and large delocalized anions, is in accordance with its molecular characteristics, that of a flexible linear molecule possessing a large number of electron-donating ester oxygens suitably spaced to replace the hydration shell of cations. In turn, PHB may be rendered soluble in aqueous media by complexation to water-soluble proteins, such as serum lipoproteins and albumin. Such solvates are highly resistant to hydrolytic enzymes. These findings suggest that the physiological roles of this unique biopolymer may include the solvation of salts of polymeric anions to facilitate their movement through hydrophobic barriers, and the protection of cellular polymers from enzymatic degradation.


Poly-beta-hydroxybutyrate in staphylococci.

Staphylococci--chemoorganotrophic bacteria whose main habitats are human and animal organisms--can accumulate poly-beta-hydroxybutyrate (PHB) in their cells. The polymer is metabolized in endogenous turnovers preceding degradation of aminoacids, proteins and RNA. PHB depolymerase was not found in staphylococci but beta-hydroxybutyrate dehydrogenase was estimated, purified and characterized.


Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria.

The current knowledge on the structure and on the organization of polyhydroxyalkanoic acid (PHA)-biosynthetic genes from a wide range of different bacteria, which rely on different pathways for biosynthesis of this storage polyesters, is provided. Molecular data will be shown for genes of Alcaligenes eutrophus, purple non-sulfur bacteria, such as Rhodospirillum rubrum, purple sulfur bacteria, such as Chromatium vinosum, pseudomonads belonging to rRNA homology group I, such as Pseudomonas aeruginosa, Methylobacterium extorquens, and for the Gram-positive bacterium Rhodococcus ruber. Three different types of PHA synthases can be distinguished with respect to their substrate specificity and structure. Strategies for the cloning of PHA synthase structural genes will be outlined which are based on the knowledge of conserved regions of PHA synthase structural genes and of the PHA-biosynthetic routes in bacteria as well as on the heterologous expression of these genes and on the availability of mutants impaired in the accumulation of PHA. In addition, a terminology for the designation of PHAs and of proteins and genes relevant for the metabolism of PHA is suggested.


Polyhydroxyalkanoate production in recombinant Escherichia coli.

The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-beta-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-3HV).


Microbial degradation of natural and of new synthetic polymers.

In landfills, deposited waste material is usually faced with strictly anoxic conditions. This means that the design of new biodegradable polymers must take into consideration that degradation should be possible especially in the absence of molecular oxygen. Poly-beta-hydroxybutyrate is depolymerized by the anaerobic fermenting bacterium Ilyobacter delafieldii through an extracellular hydrolase. Monomers are degraded inside the cells through classical beta-oxidation. Polyalkanoates containing odd-numbered or branched-chain acid monomers should he degraded in an analogous manner; in most cases the final mineralization of these residues requires special pathways. A comparison of the chemistry of natural polymer biodegradation leads to the conclusion that synthetic biodegradable polymers should be designed in the future to contain linkages which can be cleaved by extracellular hydrolytic enzymes. Recent findings on aerobic and anaerobic bacterial degradation of synthetic polyethers suggest that natural evolution of new depolymerizing enzymes, perhaps from existing hydrolases, could be possible in a reasonable amount of time, provided that the monomers are likely energy sources for a broad variety of microbes.


Biodegradation of polyhydroxyalkanoates.

Degradation of poly(3-hydroxybutyrate) and copolymers with 3-hydroxyvaleric acid was investigated in natural environments, and the microorganisms involved were isolated and identified. The influence of abiotic and biotic factors on the degradation is discussed.


Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi.

The current problems with decreasing fossile resources and increasing environmental pollution by petrochemical-based plastics have stimulated investigations to find biosynthetic materials which are also biodegradable. Bacterial reserve materials such as polyhydroxyalkanoates (PHA) have been discovered to possess thermoplastic properties and can be synthesized from renewable resources. Poly-beta-hydroxybutyric acid (PHB) is at present the most promising PHA; and BIOPOL, its copolymer with poly-beta-hydroxy-valerate (PHV), is already industrially produced (ICI, UK), and used as packaging material (WELLA, FRG). According to the literature, PHA degradation has so far mainly been observed in bacteria; only under certain environmental conditions has fungal degradation of PHAs been indicated. Since fungi constitute an important part of microbial populations participating in degradation processes, a simple screening method for fungal degradation of BIOPOL, a PHA-based plastic, was developed. Several media with about 150 fungal strains from different terrestrial environments and belonging to different systematic and ecological groups were used. PHA depolymerization was tested on three PHB-based media, each with 0.1% BIOPOL or PHB homopolymer causing turbidity of the medium. The media contained either a comparatively low or high content of organic carbon (beside PHA) or were based on mineral medium with PHA as the principal source of carbon. The degradation activity was detectable due to formation of a clear halo around the colony (Petri plates) or a clear zone under the colony (test tubes).(ABSTRACT TRUNCATED AT 250 WORDS)


Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M.

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.


Biodegradability of polymers in the environment: complexities and significance of definitions and measurements.

There is a world-wide research effort to develop biodegradable polymers as a waste-management option for polymers in the environment. This effort may prove to be fruitless unless we can agree on a definition and test protocols--what do we expect biodegradable polymers to do in the environment and how do we demonstrate that they do what we expect? Establishing a definition and test protocols is not trivial; the task is made complex by the wide range of disciplines involved directly in or interested in the subject, including polymer scientists, biochemists, environmentalists, legislators, and laypeople, all with their own perspectives and expectations. In this paper, I present arguments in favor of biodegradable polymers and indicate what remains to be done to satisfy detractors that they represent a viable option for polymer waste-management.


Unidentified multiple congenital abnormalities in twins. A population-based Hungarian study.

Of 1038 index patients with multiple congenital abnormalities, 34 were twins. This 3.3 per cent is higher than the Hungarian birth rate of about 2.1 per cent. However, after the exclusion of cases with congenital abnormality association of low birth weight newborn infants and with genital anomalies of the male, the twin birth rate was 1.8 per cent. Thus, the unidentified multiple congenital abnormalities have no common cause with twinning.


Effect of thyroxine administration on renal functions in newborn infants with perinatal asphyxia.

The study was undertaken to assess the influence of thyroxine given to improve respiratory adaptation in asphyxiated neonates on the recovery of compromised renal functions. Two groups of infants with perinatal asphyxia were selected for the study. Group I consisted of 8 infants treated conventionally, while Group II included 7 infants who in addition to standard therapy were administered 50 micrograms thyroxine at admission and repeated 24 hours later. Their respective mean gestational ages were 38.7 weeks (range: 34-42 weeks) and 37.4 weeks (range: 34-41 weeks). The studies were performed on days 1, 7 and 14 and the results compared to those obtained in 13 healthy neonates with the gestational age of 39.2 weeks (range: 38-41 weeks) (Group III). Asphyxiated neonates had significantly higher plasma uric acid, xanthine, hypoxanthine and creatinine levels (p < 0.05), while their GFR proved to be markedly reduced (p < 0.01) when compared to the values of healthy controls. Moreover, there was a significant elevation of urinary excretion of NAGA (p < 0.001), urine osmolality (p < 0.05), PENa, FECa, RFI (p < 0.05) in infants presenting with perinatal asphyxia. Renal tubular responsiveness to aldosterone measured as TTKG was also found to be depressed (p < 0.025). In response to thyroxine therapy renal functional recovery appeared to be accelerated as indicated by the lower plasma creatinine level, lower rate of fractional electrolyte and urinary NAGA excretion and improved reactivity to aldosterone on days 7 and/or 14 as compared to those obtained in neonates presenting with asphyxia but without thyroxine therapy. The results seem to suggest that thyroid hormones may have an important role in the recovery of renal functions in newborn infants suffering from perinatal asphyxia.


Bicarnesine-treated carnitine deficient myopathy: clinico-chemical investigations.

Authors report on a Bicarnesine replacement therapy in an infant girl patient suffering from carnitine deficient myopathy diagnosed at 1 year of age. The hypotonic patient's motoric functions improved and she became able to walk as a result of therapy applied, but the pathological process generalized to encephalomyopathy. Free and esterified carnitine were determined from the serum and muscle biopsymaterial. After the Bicarnesine-supplementation the serum carnitine fractions elevated.


Decrease in the antioxidant capacity of red blood cells in children with celiac disease.

The erythrocyte glutathione metabolism of 11 children with acute celiac disease (CD), 11 children under gluten free diet with CD and 5 children with cow's milk allergy was compared to that of 11 children with nutritive iron deficiency and to 22 healthy children as controls. Erythrocyte glutathione (GSH) content of celiac children was elevated and the glutathione disulfide (GSSG) level was significantly decreased as compared to normal controls. Erythrocyte GSSG/GSH ratio in acute CD differed also from the one in iron deficiency. In vitro oxidative load of acetylphenylhydrazine proved the impaired glutathione stability of the erythrocytes in acute CD and cow's milk allergy. A parallel rise of methemoglobin and hemichrome level of blood cells was seen. Further on, the selenium content of the red blood cells of CD patients decreased. All alterations of the erythrocyte tended to normalize during the dietetic period. These data suggest a reduced protective capacity of erythrocytes in CD and in cow's milk allergy in childhood against oxidizing stresses.


Adolescent mothers: do they breast-feed less?

Two groups of mothers with infants between sixty and eighty-nine days of age, from the same Health Area of the city of Havana were studied. The first group's age was nineteen years or less and members of the second one were twenty to twenty-five years old. The sociocultural level of the older group was significantly more favourable. The proportion of mothers exclusively breast-feeding was lower among the teenagers, and the frequency of complete weaning by the end of the third month was higher in their group. Duration of breast-feeding, either exclusive or combined with bottle-feeding was also shorter in the group of adolescent mothers, and frequency of diarrheal episodes was higher among their infants; body weight for age and body weight for length showed a shift towards lower percentile channels in these infants. Such differences were evident despite of that both groups of mothers had the same opportunity to receive information about the advantages of breast-feeding and there were no differences in the frequency and quality of prenatal care.


Minimizing false positive diagnoses in newborn screening for galactosemia.

Heat and humidity, rather than summertime heat alone, cause extensive loss of galactose-1-phosphate uridyltransferase activity in mailed blood spots and seem to account for false positive diagnoses of galactosemia. The spots are partially protected from the effects of atmospheric humidity if they are allowed to dry and then are sealed in a plastic freezer bag before being exposed to higher humidity. Conversely, extensive loss of transferase activity occurs if the samples are sealed in the bags before the spots are dry. The fluorescence from transferase activity can be monitored with greater sensitivity fluorometrically than visually. A simultaneous fluorometric determination of phosphoglucomutase activity reveals whether decreased transferase activity represents sample deterioration or galactosemia. Mg2+ and a sulfhydryl agent, such as dithiothreitol, are needed for activity of phosphoglucomutase, an enzyme in the sequence leading to the fluorescent substance (NADPH). They must be added in certain modifications of the assay.


Purification and characterization of glutathione transferase from psoriatic skin.

The glutathione transferases (GSTs) comprise a family of enzymes that catalyze the conjugation of glutathione with certain hydrophobic compounds, bind various hydrophobic compounds, and have selenium-independent glutathione peroxidase activity. Of the four classes of GST, the pi class is the only one present in keratinocytes, and pi-class GST is elevated in psoriatic epidermis. We have purified and characterized GST from psoriatic scales. Immunoreactivity was observed with pi class antisera, and amino terminal sequencing showed identity with GST from human placenta and cultured human keratinocytes. We conclude that the majority of GST activity in psoriatic skin is due to a pi-class isoenzyme, and pi-class GST may represent an index for hyperproliferation.


Analysis by fast atom bombardment mass spectrometry of phospholipids from tubuli, glomeruli, and urine of normal rats and rats with acute renal failure.

Fast atom bombardment mass spectrometry was used to characterize phospholipids from tubuli and glomeruli of normal rats and rats with acute renal failure. It was possible to assess the molecular species of the principal phospholipidic classes. In all of them, the most abundant species contained a residue of arachidonic acid. The phospholipids of urine were also analyzed, showing the presence of the major molecular species of several phospholipid classes. Excretion of phospholipids was greater in urine from rats with acute renal failure.


The effect of drug exposure on thyroid hormone levels of newborns.

This study examines the effect of women's alcohol abuse during pregnancy on the thyroid-stimulating hormone (TSH) and thyroxine (T4) levels of their babies at birth. Forty-seven babies were born to mothers identified as substance abusers. Maternal use of marijuana and/or cocaine did not result in newborns' TSH and T4 hormone levels being significantly different from non-substance-abusing controls' TSH and T4 levels. Alcohol use, however, was significantly linked to lower newborn TSH levels, lower than TSH levels of a control sample of newborns whose mothers used marijuana and/or cocaine, and lower than a control sample of newborns whose mothers abused no substances. This finding suggests further investigation of possible links between thyroid functioning and fetal alcohol syndrome (FAS).


Pyrimidine nucleotide synthesis in the rat mammary gland: changes in the lactation cycle and effects of diabetes.

Measurements have been made of the activities of the enzymes of the de novo and salvage pathways of pyrimidine synthesis (carbamoyl phosphate synthetase II (glutamine) (EC 6.3.5.5); dihydroorotate dehydrogenase (EC 1.3.99.11); the overall activity of Complex II (orotate phosphoribosyl pyrophosphate transferase (EC 2.4.2.10) and orotidine 5-phosphate decarboxylase (EC 4.1.1.23); uracil phosphoribosyltransferase (EC 2.4.2.9)) in the mammary gland of rats at different stages of the lactation cycle and the effects of diabetes on the activity of these enzymes in lactation have been studied. From a consideration of the changes in enzyme activities and the changes in the tissue concentration of phosphoribosyl pyrophosphate, an activator of the de novo pathway and substrate for both the de novo and salvage routes, it is concluded that the de novo pathway is the major route of pyrimidine synthesis in mammary tissue. Diabetes decreases the activity of the enzymes of the de novo pathway; the effects are particularly marked for Complex II. The present results on pyrimidine synthesis are compared to the pattern for purine synthesis previously published.


Parenchymal organ cystine depletion with long-term cysteamine therapy.

Nephropathic cystinosis is a lysosomal storage disorder characterized by renal failure, multisystem organ damage, and poor growth. Oral cysteamine therapy retards renal deterioration and enhances growth, but parenchymal organ cystine depletion has never been documented. We measured skeletal muscle cystine in 11 cystinosis patients not treated with cysteamine; analysis of their values plus 11 published values showed that muscle cystine increases linearly with age in cystinosis patients (slope, 0.074 nmol half-cystine/mg wet wt/year). In contrast, 15 patients treated for 4 to 11 years with oral cysteamine had a relatively constant muscle cystine content (slope, 0.004 nmol half-cystine/mg wet wt/year). The treated patients' mean muscle cystine, 0.091 +/- 0.064 (SD) nmol half-cystine/mg wet wt, was significantly less (P < 0.001) than that for the 11 youngest untreated patients, 0.754 +/- 0.534 nmol half-cystine/mg wet wt. On postmortem examination, a 9-year-old cystinosis patient treated for 8 years with oral cysteamine had liver, kidney, pancreas, lung, and spleen cystine values 5 to 90 times lower than those of an untreated age-matched control. We conclude that long-term oral cysteamine therapy routinely depletes cystinotic skeletal muscle of cystine; cysteamine is the treatment of choice for the prevention of both renal and nonrenal complications of cystinosis.


PCR-SSCP: a method for detection of mutations.

PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) analysis is one of the simplest and perhaps one of the most sensitive methods for detection of mutations based on PCR technology. The principles of PCR-SSCP, guidelines for experiments, and applications of this technique in various fields are reviewed.


Creation of a yeast artificial chromosome fragmentation vector based on lysine-2.

Yeast artificial chromosome (YAC) fragmentation vectors have proven to be useful reagents for mapping and modifying YAC clones through homologous recombination. Such vectors can be employed to remove a noncontiguous sequence present in chimeric YAC clones. However, previous fragmentation vectors have been based on auxotrophies not present in the background of most recombinant YAC libraries, which therefore makes them difficult to use. We have constructed an acentric YAC deletion vector pBCL that is fully compatible with AB 1380, the yeast host used in the majority of human total genomic YAC libraries. Transformation of three unrelated YACs with linearized pBCL yielded derivative clones with the expected phenotype at rates of 27%-49%. Analysis of a random cohort of these clones by pulsed-field gel electrophoresis and Southern blotting revealed single deleted structures in 31 of 34 clones. Using this vector, a nested set of deletion derivatives can efficiently and reliably be generated from human recombinant YAC clones.


A plasmid that improves the efficiency of foreign gene expression by intracellular T7 RNA polymerase.

To facilitate the construction of recombinant plasmids for expressing cloned genes with T7 RNA polymerase supplied by recombinant vaccinia virus, a plasmid expression vector was designed by combining parts of plasmids pTZ18R, pBluescript II KS+, and pAR2529. The 3043-bp plasmid pTF1 has a T7 RNA polymerase promoter, multiple cloning site for insertion of foreign genes, and a T7-specific transcription termination signal. Plasmid pTF1 had several advantages compared with the reference plasmid pAR2529, including more efficient replication in bacteria, greater flexibility in the insertion and subcloning of foreign genes, and increased efficiency of liposome-mediated introduction into cultured cells for expression of the foreign gene.


Affinity capture electrophoresis for sequence-specific DNA purification.

A new method, affinity capture electrophoresis (ACE), has been developed for the sequence-specific isolation of DNA. The target DNA is complexed with a biotinylated probe and electrophoresed in a gel equipped with a trap of immobilized streptavidin. This selectively captures the target molecule and its biotinylated probe, while other nontarget molecules pass through the trap. The target DNA is subsequently recovered from the trap by destroying the interaction between the target DNA and the biotinylated probe. Two variations of this technique, one using triple-helix formation and the other using hybridization with a uracil-containing DNA probe at the end of the target fragment, proved effective in model experiments. Since this technique requires no denaturation and handles DNA inside an agarose gel matrix, it is, in principle, applicable to the isolation of very large DNAs.


[Purine catabolites and pial arteriolar diameter in transient cerebral ischemia].

In transient cerebral ischemia, extracellular purine catabolites and pial arteriolar diameter were measured continuously. Ischemia during one hour was induced by unilateral occlusion of left middle cerebral artery in feline. Extracellular purine catabolites were sampled by in vivo brain microdialysis technique from the gray matter at ectosylvian gyrus. These catabolites were analyzed by HPLC system. Simultaneously, reactivity of pial arteriole was observed and its diameter was measured through the cranial window using intravital microscope and width analyzer. Extracellular concentrations of adenosine, inosine, hypoxanthine and xanthine were found to be 0.80 +/- 0.16 microM, 2.01 +/- 0.95 microM, 4.01 +/- 2.73 microM and 3.93 +/- 2.39 microM, respectively. During ischemia, the concentration of adenosine increased 8.7-fold and arteriolar diameter was 170% of the resting state. These findings in extracellular adenosine concentration and pial arteriolar diameter during ischemia support a role of adenosine in regulation of cerebral blood flow. After reperfusion, arteriolar diameter had returned to 120% of the resting state. But 50 min after reperfusion, pial arteriole began to dilate again coincident with the peak of xanthine concentration. These results suggest that free radicals were produced and could affect pial arterioles 50 min after reperfusion.


[The motility of IL-2 activated lymphocytes into malignant glioma].

The effects of glioma culture supernatant (GCS) and interleukin-2(IL-2) on the motility of autologous stimulated lymphocytes (ASL) were studied by using collagen gel system, chemotaxic chamber system and flow cytometric analysis. GCS inhibited the migration of ASL into collagen gel. It enhanced the ASL motility and the expression of CD 26 antigen on the cell surface. IL-2 inhibited the migration of ASL into collagen gel and had no influence on the motility of ASL, but enhanced the expression of CD 26 antigen. On the other hand, a clinical glioma specimen showed limited depth of ASL migration when injected into the tumor cavity in addition to the formation of fibrin like membrane on its surface and a layer of degenerated ASL under it. To make ASL therapy more effective to malignant glioma, the following measures should be recommended; 1) inject adequate volume of IL-2 into tumor cavity, 2) reduce the frequency of ASL administration, 3) wash out of glioma secreting factors, degenerated ASL and glioma cells in addition to reduce the volume of tumor tissue.


[Proliferation kinetics of human brain tumors by in situ double labeling with bromodeoxyuridine and iododeoxyuridine].

Nineteen patients with human brain tumors (9 gliomas, 6 metastatic brain tumors, 3 meningiomas, 1 neurinoma) received intravenous infusions of iododeoxyuridine (IUdR) and bromodeoxyuridine (BUdR) at different time sequences, to estimate the duration of S-phase (Ts) and the potential doubling time (Tp) of individual tumors. Excised tumor specimens were reacted with Br-3, a monoclonal antibody that identifies only BUdR, and with IU-4, a monoclonal antibody that recognizes both IUdR and BUdR, and then were stained immunohistochemically. The BUdR LIs varied from 0.9% to 26.0%, reflecting the malignancy of each tumor. Despite the difference in LIs, however, the Ts measured was fairly uniform. The Ts was 8.9 +/- 1.8 hrs (mean +/- SD) in malignant gliomas, 9.2 +/- 2.5 hrs (mean +/- SD) in metastatic brain tumors and 9.2 +/- 0. 3 hrs (mean +/- SD) in meningiomas, respectively. In contrast, the Tp varied from 1.3 to 12.4 days in malignant gliomas and from 1.2 to 4.4 days in metastatic brain tumors. Double logarithmic regression analysis showed a close correlation between the BUdR LI and Tp in human brain tumors with LI > 1%. Double labeling studies with BUdR and IUdR allow some of the proliferation characteristics to be determined from a single biopsy specimen and provide more useful information of each tumors than can be obtained by single-labeling studies with BUdR.


[Recurrent cases of chronic subdural hematoma--its clinical review and serial CT findings].

Eighty-eight cases (114 hematomas) of chronic subdural hematoma (SDH) were treated surgically using irrigation with or without drainage. 13 cases (14.8%), 13 hematomas (11.4%) showed recurrence after the 1st operation. They were compared with non-recurrent cases using clinical reviews and serial CT findings. The result of the study showed that all of the patients in whom recurrence occurred were male and elderly (mean age 70.4 years). Many of these recurrences occurred in cases of bilateral SDH, on the left hematoma side, and at intervals within 7 days from the onset of clinical symptoms after the 1st operation. All recurrent cases were treated surgically using irrigation without drainage. In preoperative CT findings, it was shown that high density areas and small-sized hematomas were detected at a slightly higher rate in recurrent cases than in non recurrent cases. In postoperative CT findings, it was shown that many of the recurrent patients deteriorated during an interval from 2 weeks to 2 months. It was shown that non-recurrent patients had become almost normal by 3 months after the operation. Residual air volume into the subdural space within 7 days in recurrent cases was greater than in non-recurrent cases. Therefore, using irrigation with drainage, planning the operative timing, and reduction of residual air volume into subdural space are proposed as suitable means to avoid recurrence of SDH. Patients of SDH need postoperative follow-up during an interval up to 3 months.


[Analysis of intracranial pressure pulse wave in experimental hydrocephalus].

Much has been written about the relationship between the pulse pressure (PP) of the intracranial pressure pulse wave (ICPPW) and ventricular dilatation. Some data suggest that high PP is the cause of ventricular dilatation, and other authors have reported that high amplitude of PP results from decreased intracranial compliance. In order to clarify these points, the amplitude of PP and Pressure-Volume Response (PVR: an indicator of intracranial compliance) were measured in bilateral ventricles using Howchwald's hydrocephalic model (right-left difference in ventricle size is clear, due to hemicraniectomy). Hydrocephalus was developed by means of intracisternal injection of a Kaolin powder solution using dogs. The mean ICP, amplitude of the PP, PVR and ventricular size (studied by MR image) were evaluated under pathological conditions induced by the following procedures. Group A: control. Kolin induced hydrocephalus without craniectomy. Group B: Kaolin induced hydrocephalus with right side craniectomy. Group C: Kaolin induced hydrocephalus with right side craniectomy and dural resection. Group D: Kaolin induced hydrocephalus with right side craniectomy, dural resection and temporal muscle resection. Using MR imaging, the same degree of symmetrical ventricle dilatation were identified in all groups except Group D. Group D alone demonstrated the difference of ventricular size (craniectomy side > non craniectomy side). There was no appreciable difference in mean ICP between each group. However the amplitude of PP and the PVR decreased stepwise from Group A to Group D. The difference of the amplitude of the PP and PVR between the right and left ventricles in each group was not significant. Even in the larger ventricle side (right) of Group D, the amplitude of PP was same as that of the left ventricle, and much smaller than that of other groups. The results of our research suggest that: 1) There was no relation between the ventricular dilatation and the amplitude of PP. This means that the increased amplitude of PP was not the cause of the ventricular dilatation in this model. 2) These evidences suggest that a high degree of correlation exists between the amplitude of PP and the PVR. This means that PP can be a good parameter of the intracranial compliance in this model.


[Quantitative fluorescence-immunohistochemistry of presynaptic terminals using a monoclonal antibody against a synaptic vesicle-specific protein (SVP-38) in paraffin sections].

We developed a method for investigating impairment of synaptogenesis quantitatively involving measurement of the fluorescence intensity emitted by immunohistochemically stained paraffin sections of rat brain using a monoclonal antibody (Mab 171B5) against a synaptic vesicle protein (SVP-38). We applied this method to congenitally hydrocephalic and non-hydrocephalic brains of HTX-rats, and compared the postnatal changes in the fluorescence intensity in the molecular layer of the cerebral cortex. In non-hydrocephalic HTX-rats, the fluorescence intensity remained nearly unchanged from the 1st to 7th postnatal day and then increased at an almost linear rate until the 21st postnatal day, when it reached 4.5 times the value on the 7th postnatal day. The increase thereafter was gradual until the 28th postnatal day. In hydrocephalic HTX-rats, the fluorescence intensity showed a marked reduction on the 28th postnatal day (p < 0.01). This finding indicated impairment of synaptogenesis. We believe that this method provides useful information for evaluating the impairment of synaptogenesis in various pathological conditions in mammarian brains. The basic aspects of this method which support its validity are also discussed.


[An autopsy case of acute encephalopathy associated with hypertrophy of the inferior olives].

We presented an autopsy case of a severely handicapped girl with sequelae of acute encephalopathy at 10 months old. The autopsy findings were diffuse leukomalacia in cerebral medulla, loss of nerve cells and marked gliosis in thalamus, leukomalacia with fibrous gliosis in central tegmental tracts, and marked fibrous gliosis of the cerebellar hemisphere. Purkinje cells were spared much more than granular cells. Hypertrophy of the nerve cells in the inferior olives were seen and the bundles of its efferent nerve fibers were thin. This case is worth reporting that she has suffered from brain damage about 8 years before, and the inferior olives are hypertrophic yet.


[Correlation between sequential changes in angiographical findings and clinical deteriorations during management for dural AVM of the cavernous region].

The authors treated five patients with dural arteriovenous malformation of the cavernous region (DAVM) by cervical compression procedure as the initial treatment. In four of five patients clinical symptoms were aggravated during that period. The period between the beginning of cervical compression and the deterioration of clinical symptoms ranged from 11 to 20 days. In three of four patients, the angiography examined at the time of deteriorations confirmed the same amount of A-V shunt flow and otherwise the remarkable decrease of the draining pathway compared to the findings of the angiogram performed before the management. The angiography examined at the time of the remission of the symptoms showed the increase of the draining pathway in two patients and the resolution of the DAVM in another. These findings indicate that clinical courses of the DAVM and the amount of the draining pathway correlate to each other and that cervical compression procedure may occlude the important draining pathway of the DAVM.


Functional determinants of bone structure: beyond Wolff's law of bone transformation.

Frost has suggested that functional loading controls bone mass and form as a thermostat controls temperature--by homeostatic regulation (i.e., by negative feedback). Yet, the literature contains many results that appear incompatible with the "mechanostat" hypothesis. We propose that a different type of regulation--epigenetic--is important in many aspects of bone adaptation. Epigenetic regulation, as we describe it, involves positive feedback loops and promotes differentiation as it forces elements of a system to choose between two extreme levels called attractors. Our review of bone adaptation data suggests that lamellar bone formation is regulated homeostatically, whereas the formation of woven-fibered bone or fracture callus is regulated epigenetically, that is, woven bone formation is brought about by a positive feedback loop that stimulates osteoblasts to a state of greater individual activity. This positive feedback loop may involve transforming growth factor beta (TGF beta), for which autocrine induction has been demonstrated in vivo, as well as other factors, including insulin-like growth factor (IGF) and prostaglandin E2 (PGE2). The importance of this model is that it provides a mechanism for many unexplained nonlinear events that have been observed in bone adaptation experiments. Furthermore, it provides insights into the genesis of woven bone, which is a critical step in the process of bone healing and regeneration.