Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA).

Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and HP2), therefore three genotypes are present in the population, i.e., HP1-1, HP2-1, and HP2-2. A biochemical role for nascent haptoglobin 2 (pre-haptoglobin 2 or pre-HP2) as the only known modulator of intestinal permeability has been established. In addition, elevated levels of serum pre-HP2 have been detected in multiple conditions including celiac disease and type I diabetes, which are believed to result in part through dysregulation of the intestinal barrier. In this study, we report the development of a monoclonal antibody that is specific for pre-HP2 with a binding affinity in the nanomolar range. Additional antibodies with specificities for preHP but not mature haptoglobin were also characterized. A sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated. The ELISA showed high specificity for pre-HP2 even in the presence of excess pre-HP1 or mature haptoglobins, and has excellent linearity and inter- and intra-assay reproducibility with a working range from 3.1ng/mL to 200ng/mL. Testing of sera from 76 healthy patients revealed a non-Gaussian distribution of pre-HP2 levels with a mean concentration of 221.2ng/mL (95% CI: 106.5-335.9ng/mL) and a median value of 23.9ng/mL. Compared to current approaches, this ELISA offers a validated, monoclonal-based method with high sensitivity and specificity for measuring pre-HP2 in human serum.

Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit.

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.

Clinical features and risk of recurrence among patients with vaginal intraepithelial neoplasia.

OBJECTIVE: The best treatment modality and factors affecting recurrence among women with vaginal intraepithelial neoplasia (VAIN) are yet to be determined. The aims of the current study were to describe the clinical features, results of treatment, and factors affecting recurrence among patients with VAIN. METHODS: We conducted a retrospective review of 121 women with VAIN after confirming the histologic diagnosis. Patient demographics, clinical features, and results of therapy were recorded. Factors affecting recurrence were assessed using the odds ratio and the 95% confidence intervals among patients who were followed up for 7 months or more and had at least one posttreatment Papanicolaou smear. Significant univariate odds ratios were assessed jointly in a multivariate model with a stratified analysis. RESULTS: The mean age of the patients was 35.0 (+/-17), 41% of the patients smoked, 39% had a history of human papillomavirus infection, 27% had history of sexually transmitted diseases, 22% had history of surgery for cervical intraepithelial neoplasia (CIN), and 23% had total hysterectomy. The upper third of the vagina was the most common site of VAIN and 61% of the lesions were multifocal. Associated cervical and vulvar intraepithelial neoplasia (VIN) were present in 65 and 10%, respectively. Recurrences of VAIN and progression to invasive vaginal cancer occurred in 33 and 2%, respectively. Recurrences following partial vaginectomy, laser, and 5-fluorouracil were 0, 38, and 59%, respectively (P = 0.0001). Multifocality and method of treatment were significant independent predictors of VAIN recurrences (odds ratio 3.3, 95% CI 1.2, 9.2, P = 0.02, and 22.4, 95% CI 1.3, 393.6, P = 0.001, respectively), with no interaction, based on a stratified analysis. CONCLUSIONS: VAIN occurs most often among women with CIN or VIN, commonly involves the upper third of the vagina, and is often multifocal. Partial vaginectomy provides the highest cure rate and multifocality is a risk factor for recurrence.

Preoperative differences between male and female patients with sleep apnea.

OBJECTIVES/HYPOTHESIS: To evaluate the differences between female and male patients with obstructive sleep apnea syndrome (OSAS) in the preoperative period. STUDY DESIGN: Nonrandomized cross-sectional study. METHODS: An analysis of 686 patients (111 women and 575 men) with OSAS was completed. Multivariate modeling techniques were employed to correlate gender with the preoperative respiratory disturbance index (RDI), apnea index (AI), hypopnea index (HI), body mass index (BMI), age, and initial symptoms. RESULTS: At presentation, the male patients were significantly younger and had a lower BMI and a higher RDI and AI than the female patients. For the entire OSAS population studied, the RDI increased as the BMI increased (correlation coefficient [r] = 0.35, P = <.001). For the female patients there was a weaker correlation (r = 0.21, P =.034), and in male patients there was a stronger correlation (r = 0.40, P <.001). For the entire population there was a negative correlation between age and RDI (r = -0.15, P <.001). In female patients there was a nonsignificant correlation (r = -0.09, P =.35), and in male patients the correlation was significant (r = -0.16, P <.001). There was no difference in the reporting of the number of symptoms based on gender (P =.355). Female patients noted headaches on awakening more commonly than male patients (P =.001), and male patients noted snoring (P =.014) and stopping breathing during sleep (P =.001) more often than female patients. CONCLUSIONS: The analysis demonstrated that within a surgical population sample, gender differences exist. The findings of this series were as follows: 1) Apnea severity in women was less weight-dependent than in men; (2) in men there was a significant negative correlation between age and apnea severity; and (3) female and male patients reported the same number of signs or symptoms on presentation, although certain signs and symptoms were more commonly reported based on gender. Current clinical evaluation practices must take into account this gender disparity.

Does phosphoenolpyruvate carboxykinase have a role in both amino acid and carbohydrate metabolism?

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2. The role of the enzyme in gluconeogenesis and anaplerotic reactions in a range of organisms is discussed, along with the important function in C4 and CAM photosynthesis in higher plants. In addition, new data are presented indicating that PEPCK may play a key role in amino acid metabolism. It is proposed that PEPCK is involved in the conversion of the carbon skeleton of asparagine/aspartate (oxaloacetate) to that of glutamate/glutamine (2-oxoglutarate). This metabolism is particularly important in the transport system, seeds and fruits of higher plants.

Using immunohistochemistry to study plant metabolism: the examples of its use in the localization of amino acids in plant tissues, and of phosphoenolpyruvate carboxykinase and its possible role in pH regulation.

To understand many aspects of the metabolism of complex plant structures such as leaves, fruit and roots it is important to understand how metabolic processes are compartmentalized between tissues. The aim of this article is to show how immunohistochemistry, in conjunction with biochemical and physiological studies, is useful in understanding both the function of an enzyme in a tissue and metabolic processes occurring in plant tissues. This is illustrated by two examples. Firstly, the use of immunohistochemisty in the localization of amino acids in plant tissues is described. Secondly, the use of immunohistochemistry in understanding the function of an enzyme in a tissue and the metabolic processes occurring within the tissue is described. To illustrate this the example of phosophoenolpyruvate carboxykinase (PEPCK), an enzyme which is present in many plant tissues in which its function is unknown, is used. Evidence is provided that PEPCK may play a role in pH regulation in tissues active in the metabolism of nitrogen.

Are isocitrate lyase and phosphoenolpyruvate carboxykinase involved in gluconeogenesis during senescence of barley leaves and cucumber cotyledons?

The aim of this study was to investigate whether gluconeogenesis catalysed by phosphoenolpyruvate carboxykinase (PEPCK) occurs during leaf senescence. This was addressed by determining changes in the abundance and intercellular location of enzymes necessary for gluconeogenesis during the senescence of barley leaves and cucumber cotyledons. PEPCK was never present in barley leaves, despite the presence of large amounts of isocitrate lyase (ICL), a key enzyme of the glyoxylate cycle, and of its product, glyoxylate. Although PEPCK was present in non-senescent cucumber cotyledons, its abundance declined during senescence. Throughout senescence, PEPCK was only present in the trichomes and vasculature, whereas ICL was located in mesophyll cells. Pyruvate,Pi dikinase (PPDK) which, in concert with NAD(P)-malic enzyme, is also capable of catalysing gluconeogenesis, was present in non-senescent barley leaves and cucumber cotyledons, but in both plants its abundance decreased greatly during senescence. The abundance of ICL was greatly reduced in senescing detached barley leaves by either illumination or by co-incubation with sucrose, and greatly increased in darkened attached barley leaves. These results argue against the large-scale occurrence of gluconeogenesis during senescence catalysed either by PEPCK or PPDK. In cucumber cotyledons, PEPCK may play a role in metabolic processes linked to the export of amino acids, a role in which phosphoenolpyruvate carboxylase may also be involved. The amount of ICL was increased by starvation and during senescence may function in the conversion of lipids to organic acids, which are then utilised in the mobilisation of amino acids from leaf protein.

An immunohistochemical study of the compartmentation of metabolism during the development of grape (Vitis vinifera L.) berries.

The compartmentation of key processes in sugar, organic acid and amino acid metabolism was studied during the development of the flesh and seeds of grape (Vitis vinifera L.) berries. Antibodies specific for enzymes involved in sugar (cell wall and vacuolar invertases, pyrophosphate: fructose 6-phosphate phosphotransferase, aldolase, NADP-glyceraldehyde-P dehydrogenase, cytosolic fructose 1,6-bisphosphatase), photosynthesis (Rubisco, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase), amino acid metabolism (cytosolic and mitochondrial aspartate aminotransferases, alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase), organic acid metabolism (phosphoenolpyruvate carboxylase, NAD- and NADP-dependent malic enzyme, ascorbate peroxidase), and lipid metabolism (acetyl CoA carboxylase, isocitrate lyase) were used to determine how their abundance changed during development. There were marked changes in the abundance of many of these enzymes in both the flesh and seeds. The intercellular location of some enzymes was investigated using immunohistochemistry. Several enzymes (e.g. phosphoenolpyruvate carboxylase and those involved in amino acid metabolism) were associated with tissues likely to function in the transport of imported assimilates, such as the vasculature. Although other enzymes (e.g. NADP-malic enzyme and soluble acid invertase, involved in the metabolism of sugars and organic acids) were largely present in the parenchyma cells of the flesh, their distribution was extremely heterogeneous. This study shows that when considering the metabolism of complex structures such as fruit, it is essential to consider how metabolism is compartmentalized between and within different tissues, even when they are apparently structurally homogeneous.

Early polysomnographic findings and long-term subjective results in sleep apnea patients treated with laser-assisted uvulopalatoplasty.

OBJECTIVES/HYPOTHESIS: To examine the long-term results of laser-assisted uvulopalatoplasty (LAUP) for the treatment of patients with obstructive sleep apnea syndrome (OSAS). STUDY DESIGN: One hundred eighty-two OSAS patients treated with LAUP were followed for an average of 4 years. Of these patients, 131 have completed treatment. Complete polysomnographic data are available on 48 patients. Five patients were eliminated owing to nasal surgery performed during the period between the preoperative and postoperative sleep studies, thus 43 patients were included who had only LAUP treatment. Another three patients were eliminated because their postoperative polysomnograms were performed less than 6 weeks after the last procedure. Complete long-term subjective data are available on 31 patients. METHODS: Between July 1993 and July 1998, 1038 patients were evaluated for LAUP treatment for snoring and OSAS. Of the 1038 patients, 409 were treated with LAUP and the remaining 629 were treated with other surgical procedures, devices, or behavior modification. The 40 patients who underwent only LAUP treatment for OSAS and have complete polysomnographic data are the focus of this study. RESULTS: The significant objective polysomnographic findings include a decrease in the respiratory disturbance index (RDI) from 25.0 +/- 2.8 to 15.3 +/- 2.9 (P = < .0001), and a decrease in the apnea index (AI) from 14.6 +/- 2.6 to 9.0 +/- 2.4 (P = .00248). In addition, the percentage of time spent in rapid eye movement (REM) sleep significantly increased after LAUP, from 13.3% +/- 1.0 to 17.6% +/- 1.4 (P = .0149). The long-term subjective results indicate that the presenting complaints were improved after LAUP in 74.9% +/- 4.1 with a mean follow-up of 4.04 years +/- 72 days (median, 4.63 y). CONCLUSIONS: LAUP remains a safe and effective treatment for carefully selected patients suffering from OSAS. Five years after LAUP was introduced in the United States, objective polysomnographic data demonstrate reduction in the RDI and increased time spent in REM sleep after treatment. In addition, the long-term subjective results show that the presenting symptoms were no longer present in 75% of patients treated with LAUP.

Laser-assisted uvulopalatoplasty for the treatment of mild, moderate, and severe obstructive sleep apnea.

OBJECTIVES/HYPOTHESIS: Until recently, laser-assisted uvulopalatoplasty (LAUP) has been used to treat only snoring and mild cases of obstructive sleep apnea (OSA). The purpose of this study was to evaluate the efficacy and safety of LAUP in patients with mild, moderate, and severe OSA. STUDY DESIGN: A prospective study of 38 patients who completed LAUP for the treatment of OSA who were evaluated based on the severity level of their preoperative apnea. MATERIALS AND METHODS: Between July 1993 and December 1995, 96 patients with a diagnosis of OSA based on polysomnography underwent staged outpatient LAUP treatment. Thirty-eight patients completed treatment and obtained postoperative polysomnography. Postoperative complications and polysomnographic findings were reviewed. RESULTS: Fifteen patients had a diagnosis of mild apnea, 12 had moderate apnea, and 11 had severe apnea based on preoperative polysomnography. The surgical response rates, defined as greater than or equal to 50% reduction in the postoperative respiratory disturbance index (RDI) and a postoperative RDI of less than 20, were 46.7% in the mild apneics, 41.7% in the moderate apneics, and 45.5% in the severe apneics. Postoperative complications in this series included minor bleeding, oral candidiasis, and temporary velopharyngeal insufficiency. There were no serious complications. CONCLUSIONS: In the carefully selected and prepared patient with mild, moderate, or severe OSA, LAUP should be considered a surgical option for the treatment of this disorder. LAUP remains a cost-effective and safe alternative to uvulopalatopharyngoplasty.

Guidelines for the use of radio-iodine, thyroid hormone, and treatment of metastatic disease in patients with differentiated thyroid cancer.

The treatment of metastatic differentiated thyroid carcinoma is currently at a crossroad. The stunning effect of imaging doses of RAI on subsequent treatment doses is being recognized. Alternatives to RAI imaging for diagnostic purposes are being tested; these include ultrasonography for local and cervical nodal remnants/recurrences, Sestamibi and other isotope scanning that do not require the discontinuation of TSH suppression, and the measurement of circulating thyroglobulin that is rapidly becoming the cornerstone of the detection and the treatment follow-up of metastatic carcinoma.

Comparison of 12 assays for detecting hCG and related molecules in urine samples from Down syndrome pregnancies.

Urine is a new medium for Down syndrome testing. In an effort to determine the best type of human chorionic gonadotropin (hCG)-related immunoassay for urine testing, we examined 14 Down syndrome and 91 unaffected pregnancy urine samples with 12 established assays. The assays included (a) those that detect hCG beta-core fragment only; (b) those that detect beta-core fragment with less than 18 per cent free beta-subunit cross-reactivity; (c) that which equally detects free beta-subunit and beta-core fragment; and (d) those that detect hCG, free beta-subunit, or combinations thereof. The seven type a and b assays had the highest sensitivity for Down syndrome. The median MOM for Down syndrome was 5.93 (range 4.73-7.53). At a 10 per cent false-positive rate, the median observed detection rate was 93 per cent (range 79-100 per cent) and the median predicted detection rate was 85 per cent (range 69-96 per cent). The assays that did not mainly detect beta-core fragment (types c and d) had poorer screening performance. The median MOM for Down syndrome was 2.70 (range 2.16-3.63 MOM). At a 10 per cent false-positive rate, the median observed detection rate was 50 per cent (range 36-64 per cent) and the median predicted detection rate was 37 per cent (range 21-62 per cent). We infer that the assays that only detect beta-core fragment, or beta-core fragment with minor free beta-subunit cross-reactivity (types a and b), are the better urine-based tests for Down syndrome screening.

UGP--a tumor marker of gynecologic and breast malignancies? Specificity and sensitivity in pretherapeutic patients and the influence of hormonal substitution on the expression of UGP.

Urinary gonadotropin peptide (UGP) is a 10,300 Dalton peptide which is present in the urine of pregnant women, those with trophoblast disease and those with, certain nontrophoblastic malignancies. We examined the efficiency of UGP measurement at differentiating benign from malignant gynecologic and breast diseases. UGP was measured in 1355 spot urine samples from 841 patients (343 samples from 323 healthy women and women with benign gynecologic and breast diseases, 1012 samples from 518 women with gynecologic malignant diseases or breast cancer). Using a cutoff of > 3 fmol UGP/mg urinary creatinine the specificity was 97%. The sensitivity of UGP was calculated from pretherapeutically collected samples (n = 210). The sensitivity of the test for all malignancies was 26% (ovarian malignancy (n = 27) 52%, endometrial cancer (n = 25) 32%, cervical cancer (n = 49) 29%, breast cancer (n = 72) 19%, vulvar cancer and vaginal cancer (n = 12) 17% and for carcinoma in situ of the breast or the cervix (n = 20) 0%). We also found significantly higher UGP values in postmenopausal women than in premenopausal women. Hormonal substitution significantly lowered the UGP values.

Uvulopalatopharyngoplasty versus laser-assisted uvulopalatoplasty for the treatment of obstructive sleep apnea.

Uvulopalatopharyngoplasty (UPPP) was the first surgical procedure specifically designed to address the palatal abnormalities seen in many patients with obstructive sleep apnea syndrome (OSAS). For adult apneic patients, UPPP remains the most commonly performed operation. Beginning in 1993, laser-assisted uvulopalatoplasty (LAUP) was introduced for the treatment of snoring and sleep apnea. LAUP differs from UPPP because it is performed in the office using only local anesthesia, thus being very cost-effective. Seventy-nine patients with OSAS underwent surgical treatment: 38 treated with LAUP and 41 with UPPP. The overall surgical response rate, defined as a >50% reduction in the postoperative respiratory disturbance index (RDI), was achieved in 21 (51.2%) of UPPP-treated and 18 (47.4%) of LAUP-treated patients. The RDI decreased from 30.3 +/- 3.7 to 22.2 +/- 4.4 events/h in the LAUP patients and from 52.1 +/- 5.0 to 25.5 +/- 2.7 events/h in the UPPP patients. The lowest oxyhemoglobin saturation (LS(aO2)) increased from 72.8 +/- 2.1 to 80.9 +/- 1.3% in the UPPP patients; there was no change in the LAUP patients. Postoperative complications in this series included minor bleeding, oral candidiasis, and temporary velopalatal insufficiency in both groups. In the carefully selected OSAS patient, LAUP should be considered a surgical alternative to UPPP. LAUP may actually be preferable to UPPP in certain cases because LAUP is cost-effective, does not require a general anesthetic or hospitalization, has very few associated complications, and time lost at work is minimal.

Levels of urinary beta-core fragment, total oestriol, and the ratio of the two in second-trimester screening for Down syndrome.

Levels of beta-core fragment and total oestriol in second-trimester maternal urine samples were measured in 32 Down syndrome pregnancies and 206 control pregnancies. Beta-core fragment and total oestriol values were corrected for the urinary creatinine level and expressed as multiples of the control medians (MOM). In addition, the ratio of the beta-core fragment level to the total oestriol level, without creatinine correction, was calculated, and expressed as MOM values. The median beta-core fragment, total oestriol, and ratio levels in Down syndrome cases were 5.42, 0.64, and 9.32 MOM, respectively. In the Down syndrome pregnancies, 66 per cent of the beta-core fragment levels were above the 95th centile of control levels, while 22 per cent of the total oestriol levels were below the fifth centile of control levels. In combination with maternal age, measurement of beta-core fragment and total oestriol levels in Down syndrome pregnancy resulted in an 80 per cent detection rate at a 5 per cent false-positive rate. Use of the ratio resulted in a univariate detection rate of 72 per cent. In combination with maternal age, the ratio resulted in a detection rate of 81 per cent at a 5 per cent false-positive rate. Based on this unmatched study, the measurement of a ratio of beta-core fragment to total oestriol levels, without the need for creatinine correction, may be useful in screening for fetal Down syndrome in second-trimester urine.

Objective assessment of snoring before and after laser-assisted uvulopalatoplasty.

Laser-assisted uvulopalatoplasty (LAUP) is an outpatient staged surgical procedure for the treatment of snoring. Each patient undergoes a series of procedures with the end point being patient and bed partner satisfaction in most cases. The purpose of this study was to objectively evaluate the frequency, pattern, and volume of snoring in patients prior to and following each LAUP procedure. A sonographic device, SNAP, which records oronasal respiration, was used to assess patients at home. A digital analysis of the frequency, pattern, and volume was then performed. Twenty-seven patients have been completely evaluated with this recording device. The findings demonstrate that the LAUP procedure alters the snoring sound. The maximum, average, and velum-like respiratory noise loudness all showed a statistically significant decrease when comparing the preoperative snoring to the final recording after treatment was completed. In addition, the fundamental frequency of the snoring increased significantly after each LAUP procedure. No change was seen in the snoring index following treatment. These objective results correlated well with the subjective responses of the patients and their bed partners. These findings are consistent with the hypothesis that the LAUP procedure alters snoring in a favorable manner by both objective data and subjective accounts.

Phosphorylation of phosphoenolpyruvate carboxykinase in plants. Studies in plants with C4 photosynthesis and Crassulacean acid metabolism and in germinating seeds.

We have previously shown that phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in vivo in the cotyledons of darkened cucumber seedlings and that phosphorylation is reversed by light [Walker and Leegood (1995) FEBS Lett. 362, 70-74]. In this study the molecular mass of PEPCK was estimated in a range of gluconeogenic seedlings and in leaves of C4 plants and plants with Crassulacean acid metabolism (CAM). Phosphorylation of PEPCK was studied in these plants by feeding tissues with [32P]Pi and assessing phosphorylation by SDS/PAGE and autoradiography of either total proteins or of immunoprecipitated protein. In gluconeogenic seedlings and most CAM plants PEPCK had a molecular mass of 74 kDa, whereas in C4 grasses the molecular mass of PEPCK was always smaller and varied from 67-71 kDa. In all gluconeogenic seedlings and leaves of CAM plants PEPCK was phosphorylated, but it was not phosphorylated in all species of C4 grasses studied. In CAM plants, phosphorylation of PEPCK occurred at night and dephosphorylation occurred during the day. In C4 grasses phosphorylation occurred when leaves were darkened and the enzyme was dephosphorylated following illumination, but it was only phosphorylated in those plants with larger (71 kDa) molecular mass forms of PEPCK.

Laser-assisted uvulopalatoplasty: postoperative complications.

Laser-assisted uvulopalatoplasty (LAUP) has become a widely used procedure for the treatment of snoring and mild sleep apnea in the United States. Between July 1993 and December 1994, the authors of this study prospectively evaluated 541 consecutive patients referred to their hospital for possible LAUP to treat loud disruptive snoring. Of the 541 patients, 275 patients had a total of 754 LAUP procedures. There were 26 complications (3.45%). These complications included postoperative hemorrhage in 16 patients (2.12%), local infection in 4 patients (0.53%), temporary palatal incompetence in 4 patients (0.53% , and temporary loss of taste in 2 patients (0.27%). None of the 16 patients with postoperative hemorrhage required a blood transfusion. Only 10 patients (1.3%) had hemorrhage that required medical attention; in the other patients, the bleeding stopped spontaneously. There were no cases of hypernasal speech, permanent palatal incompetence, nasopharyngeal stenosis, airway compromise, or death.