This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423-to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culturenegative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.The demonstrated pathogenicity for humans and animals of organisms belonging to the genus Acanthamoeba (17, 26), coupled with the difficulty of using morphological criteria for subgeneric identification of isolates (30,38), has stimulated a number of laboratories to pursue molecular methods for detection and identification. The objective is to develop methods that are suitable for both clinical and environmental applications. The identification of amoebic isolates should be very reliable and, at least for clinical use, the detection system should be very sensitive. Several research groups, including our own, have demonstrated the usefulness of PCR methods for detection of acanthamoebae (10,15,21,25,27,40). As few as 1 to 10 trophozoites can be detected. It also is possible to enhance detection of individual amoeba in very dilute liquid clinical samples with fluorescent in situ hybridization (FISH) (36). Several molecular approaches increase the reliability of specimen identification, but the use of DNA sequence variation appears to be the most promising. The variation is observed in restriction fragment length polymorphisms of complete or partial nuclear 18S rRNA genes (8,20,21,22), of complete mitochondrial 16S rRNA genes (7, 46), and of the complete mitochondrial genome (3,7,13,18,22,45). It also is observed in the DNA seq...

Glycogen serves as a repository of glucose in many mammalian tissues. Mice lacking this glucose reserve in muscle, heart, and several other tissues were generated by disruption of the GYS1 gene, which encodes an isoform of glycogen synthase. Crossing mice heterozygous for the GYS1 disruption resulted in a significant underrepresentation of GYS1-null mice in the offspring. Timed matings established that Mendelian inheritance was followed for up to 18.5 days postcoitum (dpc) and that ϳ90% of GYS1-null animals died soon after birth due to impaired cardiac function. Defects in cardiac development began between 11.5 and 14.5 dpc. At 18.5 dpc, the hearts were significantly smaller, with reduced ventricular chamber size and enlarged atria. Consistent with impaired cardiac function, edema, pooling of blood, and hemorrhagic liver were seen. Glycogen synthase and glycogen were undetectable in cardiac muscle and skeletal muscle from the surviving null mice, and the hearts showed normal morphology and function. Congenital heart disease is one of the most common birth defects in humans, at up to 1 in 50 live births. The results provide the first direct evidence that the ability to synthesize glycogen in cardiac muscle is critical for normal heart development and hence that its impairment could be a significant contributor to congenital heart defects.

The glucose storage polymer glycogen is generally considered to be an important source of energy for skeletal muscle contraction and a factor in exercise endurance. A genetically modified mouse model lacking muscle glycogen was used to examine whether the absence of the polysaccharide affects the ability of mice to run on a treadmill. The MGSKO mouse has the GYS1 gene, encoding the muscle isoform of glycogen synthase, disrupted so that skeletal muscle totally lacks glycogen. The morphology of the soleus and quadriceps muscles from MGSKO mice appeared normal. MGSKO-null mice, along with wild type littermates, were exercised to exhaustion. There were no significant differences in the work performed by MGSKO mice as compared with their wild type littermates. The amount of liver glycogen consumed during exercise was similar for MGSKO and wild type animals. Fasting reduced exercise endurance, and after overnight fasting, there was a trend to reduced exercise endurance for the MGSKO mice. These studies provide genetic evidence that in mice muscle glycogen is not essential for strenuous exercise and has relatively little effect on endurance.The two major repositories of glycogen, the polymeric storage form of glucose, are in the liver and skeletal muscle (1). In humans, these carbohydrate reserves are an important determinant of endurance upon sustained exercise, and muscle glycogen has long been viewed as a critical energy source during muscular activity (2-4). Depletion of muscle glycogen results in fatigue and impaired muscle performance and is a major determinant of endurance (2-5). Likewise, the ineffective utilization of muscle glycogen, as in patients with McArdle disease, leads to impaired exercise tolerance (6). In their "glycogen shunt" hypothesis, Shulman and Rothman (7) propose that glycogenolysis is the predominant source of energy for muscle contraction with glycogen acting essentially as an intermediate for blood glucose to enter glycolysis. Increasing muscle glycogen by manipulating diet and exercise regimens, a procedure termed "carbohydrate loading" or "glycogen supercompensation" (8), is adopted by endurance athletes to delay the onset of fatigue (4, 9 -11).Although the importance of adequate muscle glycogen to sustain exercise in humans has been well documented, caution is needed in extrapolating findings in rodents to humans. For instance, the amount of muscle glycogen, expressed as a fraction of body mass, is ϳ10-fold lower in mice than in humans (12, 13), whereas the corresponding values for liver glycogen are comparable (14). Thus, the relative role of these two glycogen storage depots may be different between the two species. The relative importance of muscle and liver glycogen stores as fuel sources for exercise has been studied extensively in rats (15-17). Exhaustive exercise either by treadmill running or swimming resulted in a reduction of muscle glycogen by 70 or Ͼ90%, respectively (15, 16). Both exercise methods reduced liver glycogen Ͼ90%. Using less strenuous exercise regimens, muscle g...

Some human papillomavirus (HPV) types, such as HPV 16, are clearly associated with cervical dysplasia; however, the role played by other HPV types occasionally found in dysplasia is less certain. In addition, most methods used to detect HPV in clinical specimens cannot easily distinguish among more than two or three HPV types in a single specimen. Therefore, the significance of infection with multiple HPV types is not known. To address this question, we analyzed cervicovaginal lavage specimens from three cohorts of women for HPV DNA using a PCR/reverse blot assay system that permits the detection and partial quantitation of 26 genital HPV types. As expected, 94.1% of women who had dysplasia (n = 34) and 71.4% of women who had atypical squamous cells of uncertain significance (ASCUS) (n = 21) on cytology had HPV DNA detected compared to 54.5% of age matched women with normal cytology. HPV 16 DNA was detected in 35% of dysplasia patients compared to 9% of cytologic normals (P = 0.0044). Dysplasia patients had a mean of 3.29 (range 0-10) different HPV types detected compared to 1.04 (range 0-7) HPV types among those with normal cytology (P < 0.0001). These data support a possible role for multiple HPV types in the development or progression of cervical dysplasia.

Glycogen is an important component of whole-body glucose metabolism. MGSKO mice lack skeletal muscle glycogen due to disruption of the GYS1 gene, which encodes muscle glycogen synthase. MGSKO mice were 5-10% smaller than wild-type littermates with less body fat. They have more oxidative muscle fibers and, based on the activation state of AMP-activated protein kinase, more capacity to oxidize fatty acids. Blood glucose in fed and fasted MGSKO mice was comparable to wild-type littermates. Serum insulin was lower in fed but not in fasted MGSKO animals. In a glucose tolerance test, MGSKO mice disposed of glucose more effectively than wild-type animals and had a more sustained elevation of serum insulin. This result was not explained by increased conversion to serum lactate or by enhanced storage of glucose in the liver. However, glucose infusion rate in a euglycemic-hyperinsulinemic clamp was normal in MGSKO mice despite diminished muscle glucose uptake. During the clamp, MGSKO animals accumulated significantly higher levels of liver glycogen as compared with wild-type littermates. Although disruption of the GYS1 gene negatively affects muscle glucose uptake, overall glucose tolerance is actually improved, possibly because of a role for GYS1 in tissues other than muscle. Diabetes 54: 3466 -3473, 2005 A fter a meal, glucose is distributed into various tissues of the body where it can be utilized as an energy source or stored as glycogen (1). Glycogen is a branched polymer of glucose residues connected by ␣-1,4-glycosidic linkages formed by the enzyme glycogen synthase (EC 2.4.1.11) and branchpoints formed via ␣-1,6-glycosidic linkages, introduced by the branching enzyme (EC 2.4.1.18). There are two mammalian isoforms of glycogen synthase. One, encoded by the GYS2 gene, appears to be expressed only in liver (2) while a second gene, GYS1, is expressed in skeletal and cardiac muscle as well as adipose tissue, kidney, and brain (3).Estimates of the contribution of skeletal muscle glycogen to glucose disposal after ingestion of carbohydrate vary. In humans, reports of ingested glucose conversion to muscle glycogen range from ϳ40% (4) up to 90% (5). It is widely accepted that muscle is an important site for glucose disposal and one might hypothesize that, in the absence of muscle glycogen, glucose clearance would be impaired. Consistent with this hypothesis, mutations in the GYS1 gene in humans have been implicated in certain diabetic populations with, for example, the Pro442Ala mutation resulting in decreased muscle glycogen synthase activity (6).We recently described the MGSKO mouse, in which the GYS1 gene is disrupted (7). Analysis of MGSKO mice confirmed the long-held supposition that glycogen synthase is required for glycogen synthesis since these animals were devoid of glycogen in cardiac and skeletal muscle (7). In the present study, we analyzed a number of metabolic parameters in the MGSKO mouse, including whole-body glucose metabolism, with an initial hypothesis that mice lacking the ability to synthesize muscl...

Condylomata acuminata, or genital warts, are proliferative lesions of genital epithelium caused by human papillomavirus (HPV) infection. HPV types 6 and 11 are most often detected in these lesions. Genital lesions consistent with exophytic condylomata acuminata were removed by excision biopsy from 65 patients, 41 of whom were otherwise healthy individuals (control group) and 24 of whom had conditions known to cause immunosuppression. Histologically, the majority of the lesions were typical condylomata acuminata. Three lesions removed from immunosuppressed individuals also contained foci of moderate to severe dysplasia (intraepithelial neoplasia grade II/III). A recently developed PCR and reverse blot strip assay was used to determine the specific HPV types present in the genital lesions. With a set of oligonucleotide primers based on the same primer binding regions used for the MY09 and MY11 primer pair, this PCR assay detects the presence of 27 HPV types known to infect the genital tract. All but two condylomata acuminata contained either HPV type 6 or 11. The predominant type in the lesions from control patients was HPV 6, while lesions from immunosuppressed types most often contained HPV 11. Condylomata acuminata from immunosuppressed patients contained significantly more overall HPV types than lesions from the control group. HPV types associated with an increased risk of dysplasia (high-risk types) were detected in 42 (64.6%) of the total of 65 specimens; 18 (43.9%) specimens were detected in the 41 otherwise healthy individuals, and 24 (100%) specimens were detected in the 24 immunosuppressed patients. HPV 16 was the most common high-risk type detected, found in 21 of 65 (32.3%) specimens. After HPV types 6 and 11, HPV types 53 and 54 were the most frequently detected low-risk HPV types. This study demonstrates that a high percentage of condylomata acuminata lesions contain multiple HPV types, including types associated with a high risk of dysplastic abnormalities. Further studies are needed to determine the influence these additional HPV types have on the epidemiology of genital tract HPV infections and the natural history of condylomata acuminata, especially in immunosuppressed patients.

During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis is sustained, in part, by increased expression of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increase in GA activity results from the pH-responsive stabilization of GA mRNA. The 3'-untranslated region (3'-UTR) of GA mRNA contains a direct repeat of an eight-base AU-rich element (ARE) that binds zeta-crystallin/NADPH:quinone reductase (zeta-crystallin) with high affinity and functions as a pH-response element. RNA EMSAs established that zeta-crystallin also binds to the full-length 3'-UTR of GDH mRNA. This region contains four eight-base sequences that are 88% identical to one of the two GA AREs. Direct binding assays and competition studies indicate that the two individual eight-base AREs from GA mRNA and the four individual GDH sequences bind zeta-crystallin with different affinities. Insertion of the 3'-UTR of GDH cDNA into a beta-globin expression vector (pbetaG) produced a chimeric mRNA that was stabilized when LLC-PK1-F+ cells were transferred to acidic medium. A pH-responsive stabilization was also observed using a betaG construct that contained only the single GDH4 ARE and a destabilizing element from phosphoenolpyruvate carboxykinase mRNA. Therefore, during acidosis, the pH-responsive stabilization of GDH mRNA may be accomplished by the same mechanism that affects an increase in GA mRNA.

Phosphoenolpyruvate carboxykinase (PEPCK) is regulated solely by alterations in gene expression that involve changes in rates of PEPCK mRNA transcription and degradation. A tetracycline-responsive promoter system was used to quantify the half-life of various chimeric ␤-globin-PEPCK (␤G-PCK) mRNAs in LLC-PK 1 -F ؉ cells. The control ␤G mRNA was extremely stable (t1 ⁄2 ‫؍‬ 5 days). However, ␤G-PCK-1 mRNA, which contains the entire 3-UTR of the PEPCK mRNA, was degraded with a half-life of 1.2 h. RNase H treatment indicated that rapid deadenylation occurred concomitant with degradation of the ␤G-PCK-1 mRNA. Previous studies indicate that PCK-7, a 50-nucleotide segment at the 3-end of the 3-UTR, binds an unidentified protein that may contribute to the rapid decay of the PEPCK mRNA. However, the chimeric ␤G-PCK-7 mRNA has a half-life of 17 h. Inclusion of the adjacent PCK-6 segment, a 23-bp AU-rich region, produced the ␤G-PCK-6/7 mRNA, which has a half-life of 3.6 h. The ␤G-PCK-3 mRNA that contains the 3-half of 3-UTR was degraded with the same half-life. Surprisingly, the ␤G-PCK-2 mRNA, containing the 5-end of the 3-UTR, was also degraded rapidly (t1 ⁄2 ‫؍‬ 5.4 h). RNA gel shift analyses established that AUF1 (hnRNP D) binds to the PCK-7, PCK-6, and PCK-2 segments with high affinity and specificity. Mutational analysis indicated that AUF1 binds to a UUAUUUUAU sequence within PCK-6 and the stem-loop structure and adjacent CU-region of PCK-7. Thus, AUF1 binds to multiple destabilizing elements within the 3-UTR that participate in the rapid turnover of the PEPCK mRNA.