Determination of parameters for enzyme therapy using L-asparaginase entrapped in canine erythrocytes

The antitumor agent L-asparaginase was entrapped in canine erythrocytes by a single dialysis encapsulation (efficiency mean = 30%). Concentration of asparaginase in carrier cells was about 240 IU/ml, with an average of 62% cell recovery. Use of a double dialysis procedure increased the L-asparaginase concentration within carrier cells to 530 IU/ml, with an overall cell recovery of 53.9%. In vitro efflux experiments showed L-asparaginase-loaded canine carriers were stable at both 4 and 37 degrees C for an 18-h period. In vivo cell survival studies showed that carrier cells did circulate and that L-asparaginase had a half-life of 6.5 days. No evidence suggesting that the enzyme left the cell was found. Carrier cells prepared with [3H]inulin and [14C]sucrose were stored at 4 degrees C for 2 weeks and began to show signs of deterioration after 2 days.     

Mapping sequenced E.coli genes by computer: software, strategies and examples.

Methods are presented for organizing and integrating DNA sequence data, restriction maps, and genetic maps for the same organism but from a variety of sources (databases, publications, personal communications). Proper software tools are essential for successful organization of such diverse data into an ordered, cohesive body of information, and a suite of novel software to support this endeavor is described. Though these tools automate much of the task, a variety of strategies is needed to cope with recalcitrant cases. We describe such strategies and illustrate their application with numerous examples. These strategies have allowed us to order, analyze, and display over one megabase of E. coli DNA sequence information. The integration task often exposes inconsistencies in the available data, perhaps caused by strain polymorphisms or human oversight, necessitating the application of sound biological judgment. The examples illustrate both the level of expertise required of the database curator and the knowledge gained as apparent inconsistencies are resolved. The software and mapping methods are applicable to the study of any genome for which a high resolution restriction map is available. They were developed to support a weakly coordinated sequencing effort involving many laboratories, but would also be useful for highly orchestrated sequencing projects.     

The effects of mispair and nonpair correction in hybrid DNA on base ratios (G + C content) and total amounts of DNA

Base ratios and total DNA amounts can vary substantially between and within higher taxa and genera, and even within species. Gene conversion is one of several mechanisms that could cause such changes. For base substitutions, disparity in conversion direction is accompanied by an equivalent disparity in base ratio at the heterozygous site. Disparity in the direction of gene conversion at meiosis is common and can be extreme. For transitions (which give purine [R]/pyrimidine [Y] mispairs) and for transversions giving unlike R/R and Y/Y mispairs in hybrid DNA, this disparity could give slow but systematic changes in G + C percentage. For transversions giving like R/R and Y/Y mispairs, it could change AT/TA and CG/GC ratios. From the extent of correction direction disparity, one can deduce properties of repair enzymes, such as the ability (1) to excise preferentially the purine from one mispair and the pyrimidine from the other for two different R/Y mispairs from a single heterozygous site and (2) to excise one base preferentially from unlike R/R or Y/Y mispairs. Frame-shifts usually show strong disparity in conversion direction, with preferential cutting of the nonlooped or the looped-out strand of the nonpair in heterozygous h-DNA. The opposite directions of disparity for frame-shifts and their intragenic suppressors as Ascobolus suggest that repair enzymes have a strong, systematic bias as to which strand is cut. The conversion spectra of mutations induced with different mutagens suggest that the nonlooped strand is preferentially cut, so that base additions generally convert to mutant and deletions generally convert to wild-type forms. Especially in nonfunctional or noncoding DNA, this could cause a general increase in DNA amounts. Conversion disparity, selection, mutation, and other processes interact, affecting rates of change in base ratios and total DNA.      

Effects of size and sex on the courting success and foraging behaviour of Embiotoca jacksoni

To test life-history theory that body size and sex should influence how animals allocate time to foraging versus reproductive activities, we measured the effects of size and sex on courting success and foraging behaviour of black surfperch Embiotoca jacksoni off Santa Catalina Island, southern California. Observations of focal fish were made while snorkelling, during which the length of each fish (estimated to the nearest cm), total duration of courting encounters and foraging rates were recorded. We made observations during and outside the mating season. Courtship occurred only between pairs and its duration increased with the size of both the male and female. Although males would court females that were smaller or larger than themselves, pairs that were closely matched in size had long courting sessions, whereas those that differed considerably in size courted only briefly. Small fish foraged more than larger fish, both during and outside the mating season. Males and females foraged at similar rates outside of the mating season, but during the mating season males reduced their foraging rates to less than half that seen outside of the mating season, whereas females continued to forage at the same rate. This decrease in foraging rate of males during the mating season was seen in all sizes of males but was proportionally greatest in the largest males. These observations indicate that males trade off time spent on foraging for time spent courting during the mating season, whereas females do not.     

Beneficial effects of l-arginine on reducing obesity: potential mechanisms and important implications for human health

Over the past 20 years, growing interest in the biochemistry, nutrition, and pharmacology of l-arginine has led to extensive studies to explore its nutritional and therapeutic roles in treating and preventing human metabolic disorders. Emerging evidence shows that dietary l-arginine supplementation reduces adiposity in genetically obese rats, diet-induced obese rats, finishing pigs, and obese human subjects with Type-2 diabetes mellitus. The mechanisms responsible for the beneficial effects of l-arginine are likely complex, but ultimately involve altering the balance of energy intake and expenditure in favor of fat loss or reduced growth of white adipose tissue. Recent studies indicate that l-arginine supplementation stimulates mitochondrial biogenesis and brown adipose tissue development possibly through the enhanced synthesis of cell-signaling molecules (e.g., nitric oxide, carbon monoxide, polyamines, cGMP, and cAMP) as well as the increased expression of genes that promote whole-body oxidation of energy substrates (e.g., glucose and fatty acids) Thus, l-arginine holds great promise as a safe and cost-effective nutrient to reduce adiposity, increase muscle mass, and improve the metabolic profile in animals and humans.     

Cumulative inflammatory load is associated with short leukocyte telomere length in the Health, Aging and Body Composition Study

Background

Leukocyte telomere length (LTL) is an emerging marker of biological age. Chronic inflammatory activity is commonly proposed as a promoter of biological aging in general, and of leukocyte telomere shortening in particular. In addition, senescent cells with critically short telomeres produce pro-inflammatory factors. However, in spite of the proposed causal links between inflammatory activity and LTL, there is little clinical evidence in support of their covariation and interaction.

Methodology/Principal Findings

To address this issue, we examined if individuals with high levels of the systemic inflammatory markers interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) had increased odds for short LTL. Our sample included 1,962 high-functioning adults who participated in the Health, Aging and Body Composition Study (age range: 70–79 years). Logistic regression analyses indicated that individuals with high levels of either IL-6 or TNF-α had significantly higher odds for short LTL. Furthermore, individuals with high levels of both IL-6 and TNF-α had significantly higher odds for short LTL compared with those who had neither high (OR = 0.52, CI = 0.37–0.72), only IL-6 high (OR = 0.57, CI = 0.39–0.83) or only TNF-α high (OR = 0.67, CI = 0.46–0.99), adjusting for a wide variety of established risk factors and potential confounds. In contrast, CRP was not associated with LTL.

Conclusions/Significance

Results suggest that cumulative inflammatory load, as indexed by the combination of high levels of IL-6 and TNF-α, is associated with increased odds for short LTL. In contrast, high levels of CRP were not accompanied by short LTL in this cohort of older adults. These data provide the first large-scale demonstration of links between inflammatory markers and LTL in an older population.     

Capillary electrophoresis for the investigation of prostate-specific antigen heterogeneity

Prostate-specific antigen (PSA) is a single-chain glycoprotein that is used as a biomarker for prostate-related diseases. PSA has one known posttranslational modification, a sialylated diantennary N-linked oligosaccharide attached to the asparagine residue N45. In this study capillary electrophoresis (CE) was employed to separate the isoforms of seven commercially available free PSA samples, two of which were specialized: enzymatically active PSA and noncomplexing PSA. The free PSA samples examined migrated as four to nine distinct, highly resolved peaks, indicating the presence of several isoforms differing in their oligosaccharide compositions. Overall, the use of CE provides a rapid, reproducible method for separation of PSA into its individual isoforms.     

Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis

Background

At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences.

Results

Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences.

Conclusion

High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.