Factors affecting calcium lactate and liquid expulsion defects in Cheddar cheese.

This paper summarizes the results of 2 studies designed to investigate the influence of several manufacturing and curing treatments on the appearance of Cheddar cheese defects. Specifically, 2 defects, calcium lactate crystal formation and the expulsion of free liquid (weeping) were monitored in Cheddar cheese. Both studies were conducted at a commercial cheese manufacturing facility that produces Cheddar in 18.14-kg (40-lb) blocks. In the first study we monitored cheese calcium, both total and soluble during manufacture and early curing. In the second study we measured cheese pH from 3 d through 8 mo, as well as some factors that are influenced by cheese pH. Early cheese pH (3 d to 7 d) patterns were used to select vats of cheese for retail packaging. Mild Cheddar packaged at 30 d postmanufacture and sharp Cheddar packaged at 8 mo postmanufacture from the same vats were monitored for the incidence and severity of the defects. Our results indicated that factors measured in early stages of manufacture and curing (less than 7 d) such as cheese pH at mill, lactic acid concentration, nonprotein nitrogen, and calcium (total and soluble) in cheese did not correlate with the appearance of either calcium lactate or expulsion of free liquid in packaged cheeses. Factors including pH, lactic acid concentrations, and soluble calcium measured during curing (greater than 7 d) of cheese were found to be statistically significant in the development of defects and appeared to be associated with use of specific starter culture groups. In the study, 5 different starter culture groups, each consisting of a 4-strain blend of Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis, were used to manufacture the cheeses. Cheese manufactured with one particular culture group showed no incidence of calcium lactate crystal formation or weeping during curing and shelf-life of cheeses in this study. This starter group also generated the least amount of pH change in cheese during the first month of curing. From these results we conclude that starter culture group, more than any other factor measured, played an important role in the development of calcium lactate and liquid expulsion defects in Cheddar cheese. Starter culture group appeared to strongly influence cheese pH, lactic acid, and soluble calcium concentrations during curing and storage.

Differences in organization between acute and chronic atrial fibrillation in dogs.

OBJECTIVES: The purpose of this study was to determine differences in acute and chronic atrial fibrillation (AF) "organization" in canine models. BACKGROUND: Electrophysiologic changes occur during atrial remodeling, but little is known about how remodeling affects AF organization. We hypothesized that atrial remodeling induced by long-term rapid atrial rates heterogeneously decreases AF organization. METHODS: In seven dogs, acute AF was induced by atrial burst pacing, and in eight dogs chronic AF was created by six weeks of continuous rapid atrial pacing. Atrial fibrillation was epicardially mapped from the right atria (RA) and left atria (LA). Atrial cycle length (CL), spatial organization and activation maps were compared. Spatial organization was quantified by an objective signal processing measure between multiple electrograms. RESULTS In acute AF, mean CL was slightly shorter in the LA (124 +/- 16 ms) than it was in the RA (131 +/- 14 ms) (p < 0.0001). In chronic AF, LA CL (96 +/- 14 ms) averaged 24 ms shorter than RA CL (121 +/- 18 ms) (p < 0.0001). Right atria and LA in acute AF had similar levels of organization. In chronic AF, the LA became approximately 25% more disorganized (p < 0.0001) while the RA did not change. In acute AF, a single broad wave front originating from the posterior and medial atrium dominated LA activation. In chronic AF, LA activation was more complex, sustaining multiple reentrant wavelets in the free wall and lateral appendage. CONCLUSIONS: Acute and chronic AF exhibit heterogeneous differences in CL, organization and activation patterns. The LA in chronic AF is faster and more disorganized than it is in acute AF. Differences in the models may be due to heterogeneous electrophysiologic remodeling and anatomic constraints. The design of future AF therapies may benefit by addressing the patient specific degree of atrial remodeling.

Bypass of DNA heterologies during RuvAB-mediated three- and four-strand branch migration.

During general genetic recombination and recombinational DNA repair, DNA damages and heterologies are often encountered which must be efficiently processed by the cellular recombination machinery. In RecA-mediated three-strand exchange reactions between single-stranded circular and linear duplex DNA, or four-strand exchange reactions between gapped circular and linear duplex DNA, heterologies can only be bypassed in vitro when they are short in length and are followed by homologous DNA downstream. Larger DNA inserts block RecA-mediated strand exchange, indicating that effective bypass requires other components of the recombination machinery. The RuvA and RuvB proteins of Escherichia coli form an important part of this machinery. In this work, we have analysed the ability of RuvA and RuvB to bypass large tracts of DNA heterology in both three- and four-strand exchange reactions, using recombination intermediates made by the E. coli RecA protein. Under optimal reaction conditions for RuvAB, up to 1000 bp of DNA heterology can by bypassed in three-strand reactions and 300 bp of DNA heterology can be bypassed in four-strand reactions. Whereas high concentrations of RuvB (in the absence of RuvA) can promote homologous branch migration, we find that RuvB alone is unable to catalyse heterologous bypass, indicating an essential role for both proteins in homologous recombination and recombinational DNA repair processes. Under certain conditions, the bypass of heterology is stimulated by the single-strand binding protein SSB.

Unwinding of closed circular DNA by the Escherichia coli RuvA and RuvB recombination/repair proteins.

The RuvA and RuvB proteins of Escherichia coli promote the branch migration of Holliday junctions during genetic recombination and the recombinational repair of damaged DNA. Using a topological assay that measures the underwinding of covalently closed duplex DNA, we find that RuvA and RuvB promote the transient unwinding of relaxed or supercoiled DNA. Detection of unwinding by RuvAB requires the presence of ATP and a non-hydrolysable ATP analogue (ATP gamma S), and was not observed in the presence of ATP or ATP gamma S alone. These results indicate that RuvAB catalyse the unwinding and rewinding of duplex DNA via an intermediate that can be stabilised by the presence a non-hydrolysable cofactor. At elevated concentrations of Mg2+ (12 to 30 mM), which are known to favour RuvB binding to DNA without the need for RuvA, RuvB protein alone promotes DNA unwinding. These results show that RuvB protein, an ATPase that forms hexameric ring structures that encircle the DNA, is directly responsible for the DNA unwinding activity exhibited by RuvAB. From these results, we propose that branch migration of Holliday junctions by RuvAB occurs by the passage of double-stranded DNA through the RuvAB complex, in a reaction coupled to transient DNA unwinding.

Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins.

The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA. In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay. Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP. These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.

PCR amplification and typing of the HLA DQ alpha gene in forensic samples.

The polymerase chain reaction (PCR) was used to amplify the HLA DQ alpha gene using DNA recovered from evidentiary samples. Amplified HLA DQ alpha DNA was then typed using sequence-specific oligonucleotide probes. Slight modifications of previously published DNA extraction methods improved typing success of bloodstains and semen-containing material. Evidentiary samples, consisting of 206 known bloodstains, 26 questioned bloodstains, and 123 questioned semen-containing evidentiary materials were analyzed from 96 cases previously analyzed by restriction fragment length polymorphism (RFLP) typing in the FBI Laboratory. Of the known bloodstains, 98.5% yielded DQ alpha typing results. Of the questioned samples, 102 of 149 (24/26 bloodstains and 78/123 semen-containing materials), or 68%, produced typing results. Of the 78 cases that were RFLP inclusions, 59 yielded interpretable DQ alpha results and these were all inclusions. The remaining 19 cases could not be interpreted for DQ alpha. Of the 18 RFLP exclusions, eleven were DQ alpha exclusions, four were DQ alpha inclusions, and three could not be interpreted for DQ alpha. It is expected that because of the difference in discrimination potential of the two methods, some RFLP exclusions would be DQ alpha inclusions. Some samples that failed to produce typing results may have had insufficient DNA for analysis. Employment of a human DNA quantification method in DQ alpha casework would allow the user to more consistently use sufficient quantities of DNA for amplification. It also could provide a guide for determining if an inhibitor of PCR is present, thus suggesting the use of a procedure to improve amplification. This study provides support that the HLA DQ alpha typing procedure is valid for typing forensic samples.

The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication.

Mutants in bacterial topoisomerase (topo) IV are deficient in chromosomal partitioning. To investigate the basis of this phenotype, we examined plasmid DNA topology in conditionally lethal topo IV mutants. We found that dimeric catenated plasmids accumulated in vivo after topo IV inhibition. The catenanes were supercoiled, contained from 2 to > 32 nodes, and were the products of DNA synthesis. Electron microscopy and recombination tests proved that the catenanes have the unique structure predicted for replication intermediates. These data provide strong evidence for a model in which unlinking of the double helix can occur in two stages during DNA replication and for the critical role of topo IV in the second stage. The interlocks in the catenanes appear to be sequestered from DNA gyrase, perhaps by compartmentalization in an enzyme complex dedicated to partitioning.

Cre-lox recombination in Escherichia coli cells. Mechanistic differences from the in vitro reaction.

The mechanism of the Cre recombinase of bacteriophage P1 in Escherichia coli cells was analyzed by topological methods in order to determine the important features of the in vivo reaction. Lambda infection was used to introduce the cre gene into cells containing plasmid substrates. The products of Cre resolution on substrates with directly repeated sites were predominantly free circles, even though decatenation by DNA gyrase was blocked by the drug norfloxacin. Recombination by Cre was greatly stimulated by negative supercoiling, and inversion occurred inefficiently. These results are strikingly different from those found with purified enzyme in vitro. Our data imply that Cre recombination in vivo is much more tightly controlled than it is in vitro, and that Cre acts predominantly as a resolvase in vivo. We suggest a role for Cre-mediated recombination in P1 plasmid amplification that is consistent with the selectivity of the enzyme in vivo.

Deoxyribonucleic acid (DNA) analysis by restriction fragment length polymorphisms of blood and other body fluid stains subjected to contamination and environmental insults.

Deoxyribonucleic acid (DNA) restriction fragment length polymorphism (RFLP) profile results were obtained from bloodstains and other body fluid stains subjected to mixture with other body fluids, environmental insults (sunlight and temperature), different substrates (cotton, nylon, blue denim, glass, aluminum, and wood), and contaminants (gasoline, bleach, sodium hydroxide, soil, motor oil, detergent, phosphate salt, glacial acetic acid, and microorganisms). Of the samples that produced profile results, all had profiles that were consistent with those of untreated control samples.

Fixed-bin analysis for statistical evaluation of continuous distributions of allelic data from VNTR loci, for use in forensic comparisons.

The detection of DNA polymorphisms by RFLP analysis is having a major impact on identity testing in forensic science. At present, this approach is the best effort a forensic scientist can make to exclude an individual who has been falsely associated with an evidentiary sample found at a crime scene. When an analysis fails to exclude a suspect as a potential contributor of an evidentiary sample, a means should be provided to assess suitable weight to the putative match. Most important, the statistical analysis should not place undue weight on a genetic profile derived from an unknown sample that is attributed to an accused individual. The method must allow for limitations in conventional agarose-submarine-gel electrophoresis and Southern blotting procedure, limited sample population data, possible subpopulation differences, and potential sampling error. A conservative statistical method was developed based on arbitrarily defined fixed bins. This approach permits classification of continuous allelic data, provides for a simple and portable data-base system, and is unlikely to underestimate the frequency of occurrence of a set of alleles. This will help ensure that undue weight is not placed on a sample attributed to an accused individual.

Increased expression of a 58-kDa protein kinase leads to changes in the CHO cell cycle.

We have isolated and characterized cDNA encoding a human 58-kDa protein kinase that is homologous to the cell division control (CDC) protein kinases. This protein kinase also contains a unique N-terminal domain that may potentially regulate its function. Due to its relatedness to p34CDC2, the human p58 cDNA was overexpressed in CHO cells to determine the effect on the cell cycle. Elevated expression of p58 in these cells resulted in prolonged late telophase and early G1 phase of the cell cycle. These p58 overexpressors showed a significantly increased frequency of tubulin midbodies as well as significant increases in mitotic abnormalities. Thus, proper regulation of p58 protein kinase is essential for normal cell cycle progression in these cells.

Transient expression of a p58 protein kinase cDNA enhances mammalian glycosyltransferase activity.

The effect of expression of a p58 protein kinase on mammalian beta-1,4 galactosyltransferase enzyme activity was examined in vitro and in vivo. We found that p58 protein kinase expression enhanced galactosyltransferase enzyme activity approximately three-fold in vivo when compared to reporter gene activity. Galactosyltransferase enzyme activity was also substantially reduced in vitro when dephosphorylated, or when p58 specific antibodies were used to inhibit kinase activity. These results suggest that galactosyltransferase activity is influenced by phosphorylation, and that the p58 protein kinase may mediate this effect.

A modification of the microplate method for reverse ABO typing of bloodstains and additional validation studies.

The results of additional validation studies of a sensitive microplate hemagglutination assay for ABO reverse grouping of bloodstains are presented. The results of the validation study demonstrate the reliability of the microplate assay for use in routine serological casework. Based on these studies, the microplate assay has now replaced the Lattes crust test for ABO reverse grouping of bloodstains in the FBI Laboratory.

Inactivation of human immunodeficiency virus (HIV) by ionizing radiation in body fluids and serological evidence.

A method to use ionizing radiation to inactivate HIV (Human Immunodeficiency Virus) in human body fluids was studied in an effort to reduce the risk of accidental infection to forensic science laboratory workers. Experiments conducted indicate that an X-ray absorbed dose of 25 krad was required to completely inactivate HIV. This does not alter forensically important constituents such as enzymes and proteins in body fluids. This method of inactivation of HIV cannot be used on body fluids which will be subjected to deoxyribonucleic acid (DNA) typing.

Assembly of a polyadenylation-specific 25S ribonucleoprotein complex in vitro.

Extracts from HeLa cell nuclei assemble RNAs containing the adenovirus type 2 L3 polyadenylation site into a number of rapidly sedimenting heterodisperse complexes. Briefly treating reaction mixtures prior to sedimentation with heparin reveals a core 25S assembly formed with substrate RNA but not an inactive RNA containing a U----C mutation in the AAUAAA hexanucleotide sequence. The requirements for assembly of this heparin-stable core complex parallel those for cleavage and polyadenylation in vitro, including a functional hexanucleotide, ATP, and a uridylate-rich tract downstream of the cleavage site. The AAUAAA and a downstream U-rich element are resistant in the assembly to attack by RNase H. The poly(A) site between the two protected elements is accessible, but is attacked more slowly than in naked RNA, suggesting that a specific factor or secondary structure is located nearby. The presence of a factor bound to the AAUAAA in the complex is independently demonstrated by immunoprecipitation of a specific T1 oligonucleotide containing the element from the 25S fraction. Precipitation of this fragment from reaction mixtures is blocked by the U----C mutation. However, neither ATP nor the downstream sequence element is required for binding of this factor in the nuclear extract, suggesting that recognition of the AAUAAA is an initial event in complex assembly.

Update of the "quest" concept. Mastermind Study Club.

The mastermind study club is a unique group of dentists, whose purpose is not merely to study clinical aspects of dentistry. Rather, it is a group composed of practitioners interacting with a high level of trust and with a commitment to support each other.