Chromogranin C: A Third Component of the Acidic Proteins in Chromaffin Granules

We characterized a group of acidic proteins of bovine chromaffin granules with an antiserum raised against a protein described by Rosa and Zanini [Eur. J. Cell Biol. 31, 94-98 (1983)] in pituitary gland. In adrenal medulla the proteins reacting with this antiserum are confined to chromaffin granules. Their largest component has a Mr of 86,000 and a pI of 5.0. In addition six proteins of lower molecular weight are recognized by this antiserum. In a cell-free system only one protein is synthesized that can be precipitated with this antiserum. The properties of these proteins are very similar to those of the previously described chromogranins A and B; however, there is no immunological cross-reaction between these protein groups. We suggest this third group of acidic proteins of chromaffin granules be named chromogranins C.     

Efficacy of chlorhexidine cleansing in reducing contamination of bagged urine specimens

To determine the effectiveness of precleansing with chlorhexidine gluconate-cetrimide in reducing the contamination rate of bagged urine specimens, 62 infants admitted to a children''s hospital were randomly assigned to either receive (32 infants) or not receive (30) cleansing before bag application. Perimeatal swabs were taken before bag application and, in the treated group, after cleansing. Of the specimens from the treated group 69% were found to be contaminated, compared with 73% of those from the no-cleansing group. Chlorhexidine was ineffective in eliminating the perimeatal flora in 75% of the infants. The same organisms were present on the perimeatal swab and in the urine specimen in 95% of the infants in the treated group and 96% of those in the no-cleansing group. To estimate the contamination rate of urine specimens routinely cultured in the laboratory, 200 consecutive specimens (142 midstream and 58 bagged) were cultured. The contamination rate of the midstream urine specimens was 15%, compared with 66% for the bagged speciments. The cost of laboratory processing of contaminated bagged urine specimens at the hospital in 1983 may have been as high as $13 365. Chlorhexidine cleansing does not appear to be cost-effective. Further randomized controlled studies are needed to evaluate the effectiveness of other cleansing agents in reducing the contamination rate of bagged urine specimens.     

Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA

UDP-glucuronosyltransferase (transferase) clones were isolated from a cDNA bank constructed in pBR322 using transferase-enriched mRNA from the livers of phenobarbital-treated rats. The enrichment of mRNA was accomplished by polysome immunoadsorption with antibody to purified mouse liver transferase. This antibody was shown to bind specifically to rat transferase by Ouchterlony double diffusion analysis, immunoadsorption of glucuronidating activities, and selective inhibition of the immunoadsorption of in vitro synthesized enzyme by purified rat liver transferase. The isolated clones were verified to contain DNA complementary to transferase mRNA by hybrid translation-selection. Three classes of transferase cDNAs were characterized by restriction endonuclease mapping, and the largest insert-containing clone of each class was designated pUDPGTr-1, pUDPGTr-2, and pUDPGTr-3. Their insert sizes were approximately 2,400, 2,000, and 2,000 bp, respectively. All three cDNAs hybridized with a 2,300 +/- 150 bp mRNA, and each selected the translation of a 52,000-dalton polypeptide. Immunoadsorption of the 35S-labeled translation product could be competitively inhibited in each case by the addition of purified rat liver transferase. pUDPGTr-1 and pUDPGTr-3 inserts shared extensive sequence homology. This was demonstrated by Southern blot analysis using purified inserts and electron microscopic heteroduplex analysis. Southern blot analysis revealed that these cDNAs hybridized to overlapping genomic fragments. pUDPGTr-2 shared less sequence homology with the other two classes of cDNAs, based on the above criteria. In addition, mRNA corresponding to pUDPGTr-2 was elevated 5-fold by phenobarbital treatment, whereas the other mRNAs levels were unaffected. These studies demonstrate that in rat liver there are a minimum of three distinct transferase mRNAs, two of which may be associated with a common gene or gene family.     

Cytoskeletal disruption during human cytomegalovirus infection of human lung fibroblasts

Human cytomegalovirus (HCMV) infection causes a rapid, progressive disruption of the host cell cytoskeleton that correlates with actin depolymerization. Whole-mount (3D) electron microscopy was used to analyze the cytoskeleton of uninfected and HCMV-infected human lung fibroblast cells. Within 2 min of HCMV infection, localized areas of cytoskeletal disruption were observed. Disruption extended throughout the cytoplasm during the ensuing 45 to 90 min of infection and resulted in generalized cytoskeletal disorganization. Actin depolymerization occurred, as indicated by an increase in DNase I inhibition and alteration in the fluorescence pattern with rhodamine-conjugated phalloidin. Thus, actin appears to be the primary cytoskeletal target involved during HCMV infection. Fractionation of the virus seed inoculum showed that development of DNase I inhibitory activity in infected cells was associated only with the virus-containing fractions. Cytochalasin B treatment at early times of HCMV infection stimulated progeny virus production. This study demonstrates that rapid cytoskeletal disruption occurs during early periods of HCMV infection and indicates that actin depolymerization facilitates viral infectivity.     

UDPglucosyltransferase and its kinetic fluorimetric assay

A rapid, kinetic assay for UDPglucosyltransferase has been developed using 1-naphthol as substrate. It is based on the continuous fluorimetric monitoring of 1-naphthyl glucoside formation during the reaction at physiological pH. The conjugate is easily distinguished from aglycone, since their fluorimetric properties differ. Glucoside biosynthesis in vitro by microsomal preparations isolated from the gut and fat body of cockroaches Periplaneta americana and Leucophaea maderae, and from the green gland and hepatopancreas of the crayfish Astacus astacus, has been demonstrated. The effects of buffer, pH, MgCl2, UDP-glucuronic acid, UDP-N-acetylglucosamine, sodium cholate and sonication on the enzyme activity have been assessed. The kinetic parameters of 1-naphthol and UDP-glucose have also been determined.     

Purification and separation of tomato isolectins by chromatofocusing

Tomato lectin can be rapidly separated from a crude extract of tomato fruit proteins by chromatofocusing. The lectin is recovered from a PBE94 column in two peaks, each with a specific activity comparable to that of lectin purified by affinity chromatography on ovomucoid-Sepharose. Both isolectins consist of a single polypeptide chain (Mr 68,000) and have similar properties.     

A rapid bioluminescence method for quantifying bacterial adhesion to polystyrene

Bioluminescence ATP analysis has been used to assess bacterial adhesion with hydrophobic polystyrene tubes as the attachment surface. The assay was performed at 37 degrees C and pH 6.8 with a 10 min incubation period. A variation of more than 200-fold was observed in the adherence capacity of 34 urinary isolates of Escherichia coli, and organisms could be classified as strongly or weakly adherent. All strains capable of strong adhesion possessed both type 1 fimbriae and flagella, and maximum adhesion was expressed during the exponential growth phase. Attachment was in all cases virtually eliminated by addition of 2.5% (w/v) D-mannose to the incubation buffer. Conversely, strains which were deficient in type 1 fimbriae or flagella, or both, were weakly adherent during all phases of growth. There was no correlation between adherence of E. coli to polystyrene and adherence to buccal or uroepithelial cells, but there was a significant association with adherence to uromucoid (P less than 0.002).