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N-glycan processing and assembly defects have been demonstrated in untreated and partially treated patients with Classical Galactosaemia. These defects may contribute to the ongoing pathophysiology of this disease. The aim of this study was to develop an informative method of studying differential galactose tolerance levels and diet control in individuals with Galactosaemia, compared to the standard biochemical markers. Ten Galactosaemia adults with normal intellectual outcomes were analyzed in the study. Five subjects followed galactose liberalization, increments of 300 mg to 4000 mg/day over 16 weeks, and were compared to five adult Galactosaemia controls on a galactose restricted diet. All study subjects underwent clinical and biochemical monitoring of red blood cell galactose-1-phosphate (RBC Gal-1-P) and urinary galactitol levels. Serum N-glycans were isolated and analyzed by normal phase high-performance liquid chromatography (NP-HPLC) with galactosylation of IgG used as a specific biomarker of galactose tolerance. IgG N-glycan profiles showed consistent individual alterations in response to diet liberalization. The individual profiles were improved for all, but one study subject, at a galactose intake of 1000 mg/day, with decreases in agalactosylated (G0) and increases in digalactosylated (G2) N-glycans. We conclude that IgG N-glycan profiling is an improved method of monitoring variable galactosylation and determining individual galactose tolerance in Galactosaemia compared to the standard methods.

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In the post-genomic era, genes and proteins are now studied on a more comprehensive scale. Studying disease processes at only the genetic or transcriptomic level will give an incomplete amount of information. A proteomic approach potentially allows for a more global overview of how disease processes affect the proteins present in cells, tissues and organisms. The challenge arises in determining which proteins are affected in specific diseases and establishing which of these changes are unique to a particular disease. Existing and emerging proteomic technologies allow for high throughput analysis of proteins in a variety of sample types. Prostate cancer is a significant male health problem in the Western world. It is widely accepted that more specific prognostic and diagnostic markers of prostate cancer are urgently required. The present paper suggests that urine may be an attractive biofluid in which to pursue the identification of novel biomarkers of prostate cancer. This review introduces some proteomic techniques including mass spectrometry and the newer, quantitative proteomic strategies. It focuses on the potential application of these platforms to novel urinary biomarker identification in prostate malignancy. It also includes a synopsis of the current literature on urinary proteomics.

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We assessed the prevalence of conventional risk factors for ischaemic heart disease in patients with peripheral vascular disease, and the scope for preventative treatment with lipid-lowering therapy in this group, by retrospectively reviewing 299 patients who had undergone peripheral angiography in 1996. A total of 278 patients had severe peripheral vascular disease; 44% were current smokers at the time of their angiogram, and 36% had a history of coronary artery disease (either myocardial infarction, coronary artery bypass surgery, coronary angioplasty or angina). Cholesterol had been measured in 80 (27%) patients, of whom 26 (9%) were receiving treatment for hypercholesterolaemia. Patients with a history of ischaemic heart disease were more likely to have had their cholesterol measured (50% vs. 15%; p < 0.001). Hypertension (defined as systolic > 160 mmHg or diastolic > 90 mmHg) was present in 44%. There was no difference in the distribution of risk factors between those with and those without known ischaemic heart disease. There is a high prevalence of modifiable risk factors for coronary disease in patients with severe peripheral vascular disease. Effective prevention is available for coronary artery disease, but we found low levels of treatment. There is considerable scope for intervention to reduce the risk of coronary disease in such patients.

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Earlier studies indicated that chemically crosslinking glomerular basement membrane (GBM) rendered it more permeable to water and to macromolecules. Here possible mechanisms for the introduction of crosslinks into GBM under pathological conditions were explored. Glycation with glucose and with fructose over periods of 2 wk (fructose) and 6 weeks (glucose) rendered the GBM more permeable to water and myoglobin as judged from in vitro ultrafiltration behaviour. The membranes were also made more permeable to serum following glycation. The permeation changes were shown to be dependent on glycoxidative reactions judging by their inhibition by EDTA and DTPA. Aminoguanidine also prevented glycation from altering the permeability of GBM. Fluorescence studies indicated the formation of bityrosine in glycated GBM. Studies with oxidants showed that while hydrogen peroxide superoxide and peroxynitrite had little effect on GBM, hypochlorite anion was capable of increasing GBM permeability to water, myoglobin, albumin and serum. Changes in permeation were induced by very low quantities of hypochlorite, well within the range of the amounts of hypochlorite formed by activated neutrophils. Thus glycoxidation, or oxidation by hypochlorite, are chemical mechanisms by which GBM permeability can be increased.

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Skin burns are accepted to be a complication of defibrillation, however there is no published data on their frequency, cause and treatment. A postal questionnaire survey was designed to assess the relative frequency of defibrillation burns in coronary care units and identify the possible factors contributing to their occurrence. Treatments prescribed in coronary care units were also noted. The questionnaire was sent to the Senior Sister/Charge Nurse in all 263 coronary care units in the United Kingdom. 232 Replies were received (88.2%). Defibrillation burns were seen in 98.7% of CCU's. Ten contributory factors were proposed. The commonest implicated cause was recurrent defibrillation. The most frequently prescribed topical treatment was 1% silver sulphadiazine cream (Flamazine). Defibrillation burns are relatively common in coronary care units. Many result from recurrent defibrillation and may be unavoidable in the patient undergoing prolonged resuscitation. However there are other identifiable factors which, if avoided, may lead to a reduction in the number of burns seen.

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OBJECTIVES: To determine if cancer detection rates vary with prostate size using a sextant core biopsy pattern. METHODS: We reviewed 1021 transrectal ultrasound (TRUS)-guided sextant pattern prostate biopsies to determine if cancer detection varied based on prostate size. Prostate size was determined using a computer generated elliptical estimation method. Sextant core biopsies were taken, and the patients divided into groups based on estimated size of the prostate and biopsy outcome. Large prostates were those that were estimated by TRUS as 50 cc or more. Prostates were considered small if they were less than 50 cc. Groups were compared based on size and biopsy outcome. RESULTS: Adenocarcinoma was detected in 33% (334 of 1021) of the patients. Large prostates were noted in 34% (346 of 1021), of which 23% (80 of 346) had cancer detected by sextant biopsy. Small prostates were noted in 66% (675 of 1021), of which 38% (254 of 675) had cancer detected. The difference in cancer detection in large and small glands using a sextant pattern was statistically significant (P < 0.01). Patients with positive biopsies had significantly smaller prostate sizes (40 cc +/- 26) when compared with those with negative biopsies (51 cc +/- 33) (P < 0.01). Only 14% (8 of 58) of patients with gland sizes 100 cc or greater had positive sextant biopsies while 49% (118 of 239) with prostates 25 cc or less had cancer detected. Multivariate statistical analysis was used to control for differences in age, prostate-specific antigen (PSA), PSA density, TRUS findings, and digital rectal examination between the large and small prostate groups. The difference in cancer detection persisted (P < 0.05) CONCLUSIONS: Currently no evidence exists to support differing cancer rates based on gland size alone. Our cancer detection rate using a sextant pattern was higher in men with prostates less than 50 cc, and patients diagnosed with cancer had significantly smaller prostates than those with a negative sextant biopsy. Our data suggest that significant sampling error may occur in men with large glands, and more biopsies may be needed under these circumstances. The effects of tumor volume, focality, and specimen size in relation to overall gland size may contribute to these findings.

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Expression of bovine papillomavirus type 1 (BPV-1) late genes is limited to terminally differentiated keratinocytes in an infected epithelium. We have previously shown that although the BPV-1 late polyadenylation site is functional in nonpermissive cells, a 53-nucleotide (nt) fragment of the late 3' untranslated region acts posttranscriptionally to reduce polyadenylated cytoplasmic RNA levels. This 53-nt fragment does not appear to function by destabilizing polyadenylated cytoplasmic RNA (P. A. Furth and C. C. Baker, J. Virol. 65:5806-5812, 1991). In this study, we used site-directed mutagenesis and deletion analysis to demonstrate that the sequence AAG/GUAAGU, which is identical to the consensus 5' splice site sequence, was both necessary and sufficient for the inhibitory activity of the 53-nt fragment. Furthermore, base pairing between the 5' end of the U1 small nuclear RNA and this 5' splice site-like sequence was shown to be required for the inhibitory activity in vivo. We have also further mapped the human papillomavirus type 16 late 3' inhibitory element (I. M. Kennedy, J. K. Haddow, and J. B. Clements, J. Virol. 65:2093-2097, 1991) to a 51-nt region containing four overlapping sequence motifs with partial homology to 5' splice sites. Mutation of each of these motifs demonstrated that only one of these motifs is required for the inhibitory activity. However, the presence of the other motifs may contribute to the full inhibitory activity of the element. No BPV-1 or human papillomavirus type 16 mRNAs which are spliced by using the potential 5' splice sites present in the viral late 3' untranslated regions have been identified. This suggests that the primary function of these 5' splice site-like sequences is the inhibition of late gene expression. The most likely mechanism of action of these elements is reduction of polyadenylation efficiency, perhaps through interference with 3'-terminal exon definition.

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This study monitored a 34-yr-old distance runner for 16 wk immediately postparturition, as she trained for the 1992 United States Olympic Marathon Trials. Weight (WT), percent fat (%FAT), aerobic power (VO2max), and energy intake/expenditure were evaluated 4, 8, 12, and 16 wk post-parturition. WT declined steadily throughout the investigation, while %FAT decreased through the first 12 wk. Minimal changes in VO2max (4 wk; 52.2 ml.kg-1.min-1 to 16 wk: 55.3 ml.kg-1.min-1) occurred; however, there were substantial changes in oxygen uptake at the lactate threshold (VO2-LT) and at the onset of blood lactate accumulation (VO2-OBLA). VO2-LT increased from 35.6 ml.kg-1.min-1 at 4 wk to 43.5 ml.kg-1.min-1 at 8 wk. VO2-OBLA increased from 40.1 ml.kg-1.min-1 at 4 wk to 51.2 ml.kg-1.min-1 at 8 wk. VO2-LT and VO2-OBLA did not change during the final 8 wk of training. Energy intake was consistently below energy expenditure. No physical or medical complications were encountered during training. This subject was able to improve VO2-LT and VO2-OBLA through high-intensity training without compromising her health. The evidence indicates that well-trained female athletes, while under physician care, may participate in rigorous physical activity soon after pregnancy.

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Human cervical carcinoma cell lines that were either positive or negative for human papillomavirus (HPV) DNA sequences were analyzed for evidence of mutation of the p53 and retinoblastoma genes. Each of five HPV-positive cervical cancer cell lines expressed normal pRB and low levels of wild-type p53 proteins, which are presumed to be altered in function as a consequence of association with HPV E7 and E6 oncoproteins, respectively. In contrast, mutations were identified in the p53 and RB genes expressed in the C-33A and HT-3 cervical cancer cell lines, which lack HPV DNA sequences. Mutations in the p53 genes mapped to codon 273 and codon 245 in the C33-A and HT-3 cell lines, respectively, located in the highly conserved regions of p53, where mutations appear in a variety of human cancers. Mutations in RB occurred at splice junctions, resulting in in-frame deletions, affecting exons 13 and 20 in the HT-3 and C-33A cell lines, respectively. These mutations resulted in aberrant proteins that were not phosphorylated and were unable to complex with the adenovirus E1A oncoprotein. These results support the hypothesis that the inactivation of the normal functions of the tumor-suppressor proteins pRB and p53 are important steps in human cervical carcinogenesis, either by mutation or from complex formation with the HPV E6 and E7 oncoproteins.

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A methodologic study was performed to compare the polymerase chain reaction (PCR) and Southern blot hybridization, two commonly used testing strategies for the detection of human papillomavirus (HPV) infection. Three laboratories tested masked aliquots of exfoliated cervical cell specimens obtained from 120 women by cervicovaginal lavage. The study population included 32 women with condylomatous atypia or cervical intraepithelial neoplasia and 88 control women with no known history of cervical neoplasia. Two laboratories used PCR with different sets of consensus primers for HPV detection. The third laboratory used low-stringency Southern blot hybridization to identify all HPV types, followed by high-stringency Southern and/or dot blot hybridization to confirm specific HPV types. One of the PCR primer sets detected HPV types with a differential efficiency that was not predicted by analysis of DNA sequences or direct testing of HPV-containing plasmids. In contrast, the second PCR primer set was shown to be a much broader consensus system, detecting the same HPV types as Southern blotting, though requiring much less clinical specimen. Over 80% of women with cervical intraepithelial neoplasia or condylomatous atypia were found to be HPV infected both by Southern blotting and by the second PCR primer set. Among the control women, 11% were HPV positive by Southern blotting, while 31% were positive with the second set of primers. Most of the HPV infections found only by PCR were not due to HPV type 6, 11, 16, 18, 31, 33, or 45. These known HPV types were uncommon among normal women in the study population, even as determined by the PCR method.

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Echocardiographic left ventricular hypertrophy is thought to be helpful in grading the severity of aortic stenosis. This study compared M-mode echocardiographic left ventricular wall dimensions with Gorlin aortic valve area. Good quality echocardiograms were obtained in 294 patients with aortic stenosis who also underwent cardiac catheterization. Patients with grade 3 or 4 aortic regurgitation were excluded. The correlation was calculated between the aortic valve area and the left ventricular wall dimensions. Correlation coefficients were poor; r = 0.13 for the septum, r = 0.15 for the posterior wall, and r = 0.17 for the mean wall dimension. Correlation was not improved significantly if patients with poor left ventricular function or systemic hypertension were excluded. Correlation with other hemodynamic parameters was better, peak left ventricular systolic pressure having r values of 0.36 and 0.30 for posterior wall and septum. Mean and peak aortic valve gradient had r values approaching 0.30 for both dimensions. If the peak gradient was included in multivariate analysis, the wall dimensions then had no predictive power for severity of aortic stenosis. This study demonstrates that the degree of left ventricular wall hypertrophy is not related to the severity of aortic outflow obstruction and therefore cannot be used to grade the severity of aortic stenosis.

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In an effort to understand the physiologic processes which contribute to, or hinder the transport of stones through the ureter, we examined the intraluminal ureteral pressures and peristaltic activity above and below the acutely obstructed site. Because of patient differences, variability in stone size, shape and composition, an in vivo animal model was developed to study acute ureteral obstruction. Five adult mongrel dogs were anesthetized. A midline celiotomy was made and an open-ended ureteral catheter was inserted through a distal ureterotomy and advanced up the ureter. An angiographic balloon catheter was inserted through a small nephrotomy and directed down the ureter. The experiment was divided into phases: control, ureteral obstruction (balloon inflation) and release of obstruction (balloon deflation). Compared to control values, peristaltic rate above the obstruction increased significantly (p less than 0.05), as well as baseline, peak, and delta (peak minus baseline) pressures. In contrast, the peristaltic rate below the obstructed site remained approximately the same as its control, despite the significant decreases in baseline, peak, and delta (p less than 0.05) pressures. Failure of transmission of effective peristalsis across the obstructed site may hinder stone passage; however, this remains to be proven. Moreover, the failure of transmission of the increased rate of peristalsis past the balloon and persistence of peristaltic activity below the site of obstruction despite absence of urine flow suggest segmental forces influence peristaltic activity.

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The prevalence of right-to-left interatrial shunts was determined by contrast echocardiography in a blind comparison of 61 divers who had had decompression sickness, divided into four predetermined clinical subgroups, and a control group of 63 who had not. The prevalence of shunt was 15/63 in the controls and did not differ significantly in 24 divers with onset of neurological symptoms more than 30 minutes after surfacing (4/24) or 6 with joint pain only (1/6). In divers who had neurological symptoms within 30 minutes of surfacing the prevalence of shunt was 19/29, significantly higher. Rashes soon after surfacing were related to shunts but late rashes were not.

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The E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) encodes positive- and negative-acting factors that regulate viral gene expression. The full-length ORF encodes a transactivator, and two transcriptional repressors are expressed from the 3' half of the ORF. Previous analysis has shown that a conserved C-terminal region of 101 amino acids, which is shared by E2 transactivator and repressor proteins, contains the specific DNA binding activity. Further analysis of the E2 transactivator shows that a conserved N-terminal domain of approximately 220 amino acids is crucial for the transcriptional activation function, whereas the variable internal region is not required. The E2 proteins bind to a sequence, ACCGN4CGGT, several copies of which are sufficient to constitute an E2-dependent enhancer. By using a gel retardation assay and proteins derived by in vitro transcription and translation, we were able to show that the E2 polypeptides bind as dimers to a single DNA binding site. The dimeric E2 proteins are stable in the absence of DNA and dimerization is mediated through the DNA binding domain. This may reveal an additional mechanism of repression that could potentially result from the formation of inactive heterodimers between transactivator and repressor species.

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The long control region of bovine papillomavirus type 1 (BPV-1) can function in an orientation- and position-independent manner as an E2-dependent enhancer. Dissection of the long control region has revealed two E2-responsive elements, E2RE1 and E2RE2, which map, respectively, between nucleotides 7611 and 7806 and between nucleotides 7200 and 7386 of the BPV-1 genome. In this study, we have carried out a detailed analysis of E2RE1, which has previously been shown to be involved in the regulation of the BPV-1 promoters P89 and P7940. One characteristic of E2RE1 is the presence of a pair of ACCN6GGT motifs (E2 binding sites) at each end of the element. To determine the contribution of these sites, as well as other sequences within E2RE1, to enhancer function, specific mutations and deletions were generated by oligonucleotide reconstruction. The functional analysis of these mutations confirmed that a pair of E2 binding sites was essential for E2-dependent enhancer activity but also indicated that cooperativity between the motifs at each end of E2RE1 creates a highly responsive element. Isolated ACCN6GGT motif pairs could also act as E2-dependent enhancers but at a significantly reduced level in comparison to the intact element. The sequences between the E2 binding sites in E2RE1 were not required for enhancer function and could actually block the enhancer activity of an isolated pair of E2 binding sites when positioned between the binding sites and the enhancer-deleted simian virus 40 early promoter.

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Infection of NIH 3T3 cells with a combination of HCMV and BPV resulted in more foci than infection with BPV alone. Foci were microscopically apparent at 4 days in the mixed infection and did not appear until 2 days later in the cultures infected with BPV alone. The enhancement was abolished by heat inactivation of the HCMV and also when the HCMV was replaced by a "mock inoculum." Southern blot analysis of cellular DNA from transformed cells showed a similar amount of extrachromosomal BPV DNA in cells infected by BPV alone and in cells co-infected with HCMV. No HCMV antigens could be found in these cells by immunofluorescence. The mechanisms of the enhancement are not known. Stimulation of host DNA synthesis by HCMV could possibly increase the transforming efficiency of BPV. Alternatively, the increase in BPV transforming efficiency could be due to a transient increase in BPV-1 transcription by an HCMV transcriptional transactivation factor. Since both HCMV and human papillomaviruses are commonly found in the uterine cervix, HCMV may play a role in human cervical cancer by enhancing the carcinogenic potential of human papillomavirus.

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The effect of position in a bovine papillomavirus type 1 (BPV-1) vector on foreign gene expression was assessed with the rat preproinsulin (rI1) gene. The rI1 gene was inserted at each of the BPV-1/pML2d junctions in either transcriptional orientation in derivatives of the pdBPV-1(142-6) vector which consists of the BamHI linear genome of BPV-1 DNA cloned into pML2d. Transformed lines of C127 cells were established and assayed for rI1 gene expression. Cells containing the rI1 gene at the 3' end of the BPV-1 transforming region expressed rat proinsulin, whereas cells with the gene at the 5' end of the nontransforming region did not. Variability in the plasmid copy number or in the extent of DNA rearrangement could not account for this difference. We conclude that the expression of the rat preproinsulin gene (which is normally tissue specific for pancreatic islet cells) in C127 cells depends on the transcriptional activation afforded by viral enhancer sequences located at the 3' end of the transforming region. Intervening BPV-1 or pML2d sequences appear to block this enhancer-mediated gene activation. In agreement with enhancer-dependent activation, a rat preproinsulin gene located in a blocked position (i.e., not adjacent to the BPV-1 enhancer) could be activated by the insertion of a DNA fragment containing the simian virus 40, Moloney murine sarcoma virus, or BPV-1 enhancer element adjacent to the rI1 gene. Thus, a gene which is normally not expressed in a particular cell may be activated when placed adjacent to a viral enhancer in a BPV-1 vector.

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We constructed a mutant of bovine papillomavirus type 1 (BPV-1) DNA that lacked a transcriptional enhancer located 3' to the polyadenylation site of the early viral RNAs expressed in transformed cells. This mutant DNA, when separated from the procaryotic sequences, transforms mouse cells with an efficiency comparable to that of the full BPV-1 genome, and it exists as a stable multicopy plasmid in transformed cells. The BPV-1 distal enhancer suppresses the effects of a cis-inhibitory element in pML2 sequences but is not essential for the expression of the viral genes involved in cellular transformation or plasmid maintenance.

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