[Clinical characteristics of 272 437 patients with different histopathological subtypes of primary esophageal malignant tumors].

Objective: To characterize the histopathological subtypes and their clinicopathological parameters of gender and onset age by common, rare and sparse primary esophageal malignant tumors (PEMT). Methods: A total of 272 437 patients with PEMT were enrolled in this study, and all of the patients were received radical surgery. The clinicopathological information of the patients was obtained from the database established by the State Key Laboratory of Esophageal Cancer Prevention & Treatment from September 1973 to December 2020, which included the clinical treatment, pathological diagnosis and follow-up information of esophagus and gastric cardia cancers. All patients were diagnosed and classified by the criteria of esophageal tumor histopathological diagnosis and classification (2019) of the World Health Organization (WHO). The esophageal tumors, which were not included in the WHO classification, were analyzed separately according to the postoperative pathological diagnosis. The χ2 test was performed by the SPSS 25.0 software on count data, and the test standard α=0.05. Results: A total of 32 histopathological types were identified in the enrolled PEMT patients, of which 10 subtypes were not included in the WHO classification. According to the frequency, PEMT were divided into common (esophageal squamous cell carcinoma, ESCC, accounting for 97.1%), rare (esophageal adenocarcinoma, EAC, accounting for 2.3%) and sparse (mainly esophageal small cell carcinoma, malignant melanoma, etc., accounting for 0.6%). All the common, rare, and sparse types occurred predominantly in male patients, and the gender difference of rare type was most significant (EAC, male∶ female, 2.67∶1), followed with common type (ESCC, male∶ female, 1.78∶1) and sparse type (male∶ female, 1.71∶1). The common type (ESCC) mainly occurred in the middle thoracic segment (65.2%), while the rare type (EAC) mainly occurred in the lower thoracic segment (56.8%). Among the sparse type, malignant melanoma and malignant fibrous histiocytoma were both predominantly located in the lower thoracic segment (51.7%, 66.7%), and the others were mainly in the middle thoracic segment. Conclusion: ESCC is the most common type among the 32 histopathological types of PEMT, followed by EAC as the rare type, and esophageal small cell carcinoma and malignant melanoma as the major sparse type, and all of which are mainly occur in male patients. The common type of ESCC mainly occur in the middle thoracic segment, while the rare type of EAC mainly in the lower thoracic segment. The mainly sparse type of malignant melanoma and malignant fibrous histiocytoma predominately occur in the lower thoracic segment, and the remaining sparse types mainly occur in the middle thoracic segment.

[Sex Estimation of Typical Adult Vertebrae Morphology in Central China Based on CT Technique].

ABSTRACT: Objective The morphological data of the second thoracic vertebra and the third lumbar vertebra were measured by computerized tomography （CT）. The sex differences were analyzed and the discrimination equation was obtained. Methods The data of 274 adults （203 cases from experimental group and 69 cases from validation group） from central China were collected. Four linear data （maximum transverse length of vertebral body, maximum longitudinal length of vertebral body, maximum transverse length of vertebral foramen, maximum longitudinal length of vertebral foramen）, one angle data （angle between spinous processes） and two area （vertebral foramen area, total cross-sectional area of vertebral body） data of the second thoracic vertebra and the third lumbar vertebra were collected, respectively. Then three ratios ［maximum transverse length/　maximum longitudinal length of vertebral body, maximum transverse length/ maximum longitudinal length of vertebral foramen, vertebral foramen area/ （total cross-sectional area of vertebral body-vertebral foramen area）］ and one angle （angle between spinous processes） were obtained. The discriminant equation was established for sexual discriminant analysis. Results The morphology of the second thoracic vertebra and the third lumbar vertebra was related with gender. Four single index discriminant formulae and eleven multi-index discriminant formulae were established. The 69 validation group samples were substituted into the formula for testing, and the maximum discriminant accuracy rate of the single-index discriminant formula was 75%. The maximum discriminant accuracy rate of multi-index discriminant formula was 83%. Conclusion It is feasible to conduct individual sex analysis by the morphological indexes of second thoracic vertebra and the third lumbar vertebra. The indexes have important application values in practice.

Retrospective Analysis on 1 900 Cases of Medical Malpractices.

ABSTRACT: Objective To study the change trend, distribution characteristics and causes of medical malpractices in 18 years, through retrospective analysis of 1 900 cases of medical malpractices, in order to provide guidance for the precaution and the treatment of medical malpractices. Methods A thousand and nine hundred medical malpractice cases that were accepted by Hunan Xiangya Judical Identification Center from 2000 to 2017 were collected. Statistical analysis on the caseloads, the clients, the department distributions, the levels of the hospital and the causes of medical malpractices, etc were conducted. Results The number of cases commissioned by third-party accreditation agencies for medical fault identification was on the rise; The clients of medical malpractices changed significantly in 18 years. Most of the medical malpractice cases occurred in secondary and tertiary hospitals, significantly more in surgery, obstetrics and gynecology. The occurrence of medical malpractices was related to the doctor's insufficient prediction of the severity of the disease and the possible complications, and failure to fully inform the relevant duty of care, etc. Conclusion China's medical malpractice solutions and medical fault identification procedures are gradually improving. Strengthening the medical malpractice precaution awareness of medical workers in surgery, obstetrics and gynecology will be conducive to resolution of medical malpractices.

Stature Estimation of Sichuan Han Females Based on X-ray Photography of Total Lower Limbs.

OBJECTIVES: To establish a mathematical model of stature estimation for Sichuan Han females on the basis of the relationship between lower limbs and individual height, thus to provide evidence for forensic identification. METHODS: Samples were collected from 171 Sichuan Han females. Large flat panel multi-function digital photography system was used to take the full-body X-ray films of the lower limbs. Indexes of long bones and stature of the subjects were measured, respectively. A linear regression analysis was carried out on the correlation between them, and a mathematical model of the stature calculation was established. Then the mathematical model was used to calculate the stature of another 29 Sichuan Han females to test its accuracy. RESULTS: The maximum length of femur （x1） had the highest correlation with stature. A total of 13 linear regression equations were established （P<0.05）, with the correlation coefficient （R） 0.821-0.897 and the standard error of the estimation （SEE） 2.994-3.812 cm. The backtesting showed that the equation y=41.604+1.205 x1+1.318 x6+2.444 x12+1.852 x13-2.388 x14 had the smallest mean absolute deviation （2.485 years old） and the highest accuracy of ±2SEE （92.9%）, and that the equation y=48.783+2.568 x1 had the highest accuracy of ±1SEE （60.7%）. CONCLUSIONS: The stature estimation is high by using the long bones of the lower limbs has high accuracy.

Fracture liaison services for osteoporosis in the Asia-Pacific region: current unmet needs and systematic literature review.

The analysis aimed to identify the treatment gaps in current fracture liaison services (FLS) and to provide recommendations for best practice establishment of future FLS across the Asia-Pacific region. The findings emphasize the unmet need for the implementation of new programs and provide recommendations for the refinement of existing ones. The study's objectives were to evaluate fracture liaison service (FLS) programs in the Asia-Pacific region and provide recommendations for establishment of future FLS programs. A systematic literature review (SLR) of Medline, PubMed, EMBASE, and Cochrane Library (2000-2017 inclusive) was performed using the following keywords: osteoporosis, fractures, liaison, and service. Inclusion criteria included the following: patients ≥ 50 years with osteoporosis-related fractures; randomized controlled trials or observational studies with control groups (prospective or retrospective), pre-post, cross-sectional and economic evaluation studies. Success of direct or indirect interventions was assessed based on patients' understanding of risk, bone mineral density assessment, calcium intake, osteoporosis treatment, re-fracture rates, adherence, and mortality, in addition to cost-effectiveness. Overall, 5663 unique citations were identified and the SLR identified 159 publications, reporting 37 studies in Asia-Pacific. These studies revealed the unmet need for public health education, adequate funding, and staff resourcing, along with greater cooperation between departments and physicians. These actions can help to overcome therapeutic inertia with sufficient follow-up to ensure adherence to recommendations and compliance with treatment. The findings also emphasize the importance of primary care physicians continuing to prescribe treatment and ensure service remains convenient. These findings highlight the limited evidence supporting FLS across the Asia-Pacific region, emphasizing the unmet need for new programs and/or refinement of existing ones to improve outcomes. With the continued increase in burden of fractures in Asia-Pacific, establishment of new FLS and assessment of existing services are warranted to determine the impact of FLS for healthcare professionals, patients, family/caregivers, and society.

Nutrition therapy in esophageal cancer-Consensus statement of the Gastroenterological Society of Taiwan.

A number of clinical guidelines on nutrition therapy in cancer patients have been published by national and international societies; however, most of the reviewed data focused on gastrointestinal cancer or non-cancerous abdominal surgery. To collate the corresponding data for esophageal cancer (EC), a consensus panel was convened to aid specialists from different disciplines, who are involved in the clinical nutrition care of EC patients. The literature was searched using MEDLINE, Embase, the Cochrane Central Register of Controlled Trials, and the ISI Web of Knowledge. We searched for the best evidence pertaining to nutrition therapy in the case of EC. The panel summarized the findings in 3 sections of this consensus statement, based on which, after the diagnosis of EC, an initial distinction is made between the patients, as follows: (1) Assessment; (2) Therapy in patients with resectable disease; patients receiving chemotherapy or chemoradiotherapy prior to resection, and patients with unresectable disease, requiring chemoradiotherapy or palliative therapy; and (3) Formula. The resulting consensus statement reflects the opinions of a multidisciplinary group of experts, and a review of the current literature, and outlines the essential aspects of nutrition therapy in the case of EC. The statements are: Patients with EC are among one of the highest risk to have malnutrition. Patient generated suggestive global assessment is correlated with performance status and prognosis. Nutrition assessment for patients with EC at the diagnosis, prior to definitive therapy and change of treatment strategy are suggested and the timing interval can be two weeks during the treatment period, and one month while the patient is stable. Patients identified as high risk of malnutrition should be considered for preoperative nutritional support (tube feeding) for at least 7-10 days. Various routes for tube feedings are available after esophagectomy with similar nutrition support benefits. Limited intrathoracic anastomotic leakage postesophagectomy can be managed with intravenous antibiotics and self-expanding metal stent (SEMS) or jejunal tube. Enteral nutrition in patients receiving preoperative chemotherapy or chemoradiation provides benefits of maintaining weight, decreasing toxicity, and preventing treatment interruption. Tube feeding or SEMS can offer nutrition support in patients with unresectable esophageal cancer, but SEMS is not recommended for those with neoadjuvant chemoradiation before surgery. Enteral immunonutrition may preserve lean body mass and attenuates stress response after esophagectomy. Administration of glutamine may decrease the severity of chemotherapy induced mucositis. Enteral immunonutrition achieves greater nutrition status or maintains immune functions during concurrent chemoradiation.

The relationship between high-density lipoprotein cholesterol levels and arterial stiffness in a Taiwanese population.

BACKGROUND AND AIMS: There are few studies on the association between HDL-C levels and arterial stiffness (AS). HDL-C levels vary in males and females, and it is not clear whether the relationship between HDL-C levels and AS is influenced by gender. The purpose of this study was to investigate gender differences in the association between HDL-C levels and AS in adults. METHODS AND RESULTS: After excluding subjects using lipid-lowering agent, 7254 subjects were enrolled. The AS was assessed by measuring the brachial-ankle pulse wave velocity (baPWV) value. The quartiles of HDL-C were <38, 38-45, 45-53 and >53 mg/dL in men and <48, 48-57, 57-69 and >68 mg/dL in women, respectively. In subjects aged <50 years, none of the HDL-C quartiles were associated with baPWV values. In subjects aged ≥50 years, the highest quartile of HDL-C (beta: -37.57, 95% CI: -61.61 to -13.54) was negatively related to baPWV values. When considering gender difference in subjects aged ≥50 years, the highest quartile of HDL-C (Q4 beta: -57.22, 95% CI: -95.63 to -18.81) was inversely associated with baPWV values in women, but none of the HDL-C quartiles were related to baPWV values in men. CONCLUSIONS: A high HDL-C level was associated with a lower risk of AS in subjects aged ≥50 years in women but not in men, although this relationship was not apparent in subjects aged <50 years. The association between HDL-C level and AS is thus influenced by gender in people aged ≥50 years.

A theoretical study on photophysical properties of triphenylamine-cored molecules with naphthalimide arms and different π-conjugated bridges as organic solar cell materials.

A series of D-π-A star-shaped molecules with triphenylamine (TPA) as a core, 1,8-naphthalimide (NI) derivatives as end groups, and different π-bridges have been designed to explore their optical, electronic, and charge transport properties as organic solar cell (OSC) materials. The calculation results showed that the star-shaped molecules can lower the material band gap and extend the absorption spectrum towards longer wavelengths. The designed molecules own the longest wavelength of absorption spectra, oscillator strength, and absorption region values. Our results suggest that the designed molecules are expected to be promising candidates for OSC materials. Additionally, the molecules with ethyne, thiophene, benzo[c][1,2,5]thiadiazole (BTA), and 2,3-dihydrothieno[3,4-b][1,4]dioxine (DTD) as π-bridges and 4-pyridne, 4-aniline, and H in NI fragments have better hole- and electron transporting balance and can act as nice ambipolar materials. The values of hole mobility of molecules with ethyne as a π-bridge and NI as an end group for Pna2(1) and P2(1)/c are 5.30 × 10(-3) and 1.27 × 10(-2) cm(2) V(-1) s(-1), respectively. On the basis of the investigated results, we suggest that molecules under investigation are suitable donors of [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) and its derivatives are acceptors of solar cells.

Chronic intrathecal infusion of mibefradil, ethosuximide and nickel attenuates nerve ligation-induced pain in rats.

BACKGROUND: T-type Ca(2+) channels (TCC) are important for pain transmission, especially the Ca(V)3.2 subtype. In this study, we examined the effects of intrathecal TCC blockers in the L5/6 spinal nerve ligation pain rat model. METHODS: Under isoflurane anaesthesia, rats received right L5/6 spinal nerve ligation and intrathecal catheters (attached to an infusion pump) were sited. After surgery, saline, mibefradil, ethosuximide or NiCl2 were given intrathecally for seven days. The right hindpaw withdrawal thresholds to von Frey hair stimuli and withdrawal latencies to radiant heat were measured before and once daily for seven days after surgery. Double immunofluorescence and western blotting were used to examine the expression of Ca(V)3.2 in dorsal root ganglion (DRG) and spinal cord. RESULTS: On post-ligation day seven, rats receiving mibefradil, ethosuximide or NiCl2 had significant higher median withdrawal thresholds (15.0, 10.2, and 10.9 g) and latencies (8.0, 7.6 and 7.6 s) than saline-treated rats (1.6 g and 4.3 s, respectively). Ca(V)3.2 was expressed in parvalbumin(+), IB4(+), CGRP(+) and VR1(+) neurones in DRG and most neurones in spinal dorsal horn. Ca(V)3.2 was up-regulated in the right L5/6 DRG and spinal cord seven days after nerve ligation. CONCLUSIONS: In this study, we demonstrated that intrathecal TCC blockers attenuate the development of nerve injury-induced mechanical allodynia and thermal hyperalgesia. Our data suggest that continuous intrathecal infusion of TCC or Ca(V)3.2 blockers may be a promising alternative for the management of nerve injury-induced pain.

Identification of forensically important flesh flies based on a shorter fragment of the cytochrome oxidase subunit I gene in China.

With the development of molecular identification, there has been a great deal of discussion about the feature of the mitochondrial DNA (mtDNA) fragments. Although longer fragments may minimize stochastic variation across taxa and be more likely to reflect broader patterns of nucleotide divergence, shorter fragments have many advantages, such as quick, easy and economical. Extensive application of long mtDNA segments for species identification cannot always be achieved as a result of constraints in time and money. In the present study, a molecular identification method involving the sequencing of a 272-bp 'barcode' fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene from 55 specimens, representing 7 Chinese sarcophagid species from varying populations, was evaluated. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into seven species, which indicated the possibility of separation congeneric species with the short fragments. The results of this research will be instrumental for the implementation of the Chinese Sarcophagidae database.

Major depression during and after the menopausal transition: Study of Women's Health Across the Nation (SWAN).

BACKGROUND: It is unclear whether risk for major depression during the menopausal transition or immediately thereafter is increased relative to pre-menopause. We aimed to examine whether the odds of experiencing major depression were greater when women were peri- or post-menopausal compared to when they were pre-menopausal, independent of a history of major depression at study entry and annual measures of vasomotor symptoms (VMS), serum levels of, or changes in, estradiol (E2), follicular stimulating hormone (FSH) or testosterone (T) and relevant confounders. METHOD: Participants included the 221 African American and Caucasian women, aged 42-52 years, who were pre-menopausal at entry into the Pittsburgh site of a community-based study of menopause, the Study of Women's Health Across the Nation (SWAN). We conducted the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID) to assess diagnoses of lifetime, annual and current major depression at baseline and at annual follow-ups. Psychosocial and health factors, and blood samples for assay of reproductive hormones, were obtained annually. RESULTS: Women were two to four times more likely to experience a major depressive episode (MDE) when they were peri-menopausal or early post-menopausal. Repeated-measures logistic regression analyses showed that the effect of menopausal status was independent of history of major depression and annually measured upsetting life events, psychotropic medication use, VMS and serum levels of or changes in reproductive hormones. History of major depression was a strong predictor of major depression throughout the study. CONCLUSIONS: The risk of major depression is greater for women during and immediately after the menopausal transition than when they are pre-menopausal.

Experimental Leptospira interrogans serovar Kennewicki infection of horses.

BACKGROUND: Little information is available about experimental induction of leptospirosis in horses. OBJECTIVES: Determine serologic, hematologic responses of horses to Leptospira interrogans serovar Kennewicki infection. ANIMALS: Four adult horses seronegative for leptospirosis. METHODS: Experimental and observational study. Horses were challenged with an equine isolate of L. interrogans serovar Kennewicki at 2 different doses and different inoculation sites. After challenge, the horses were monitored for 60 days. Blood, urine, and aqueous humor samples were collected at intervals until euthanasia 60 days after infection. RESULTS: Pyrexia (39.3-40 degrees C) occurred as early as 1 day after challenge with 10x10(8)Leptospira divided equally between topical ocular and intraperitoneal injection in 2 horses. Leptospires were recovered from the blood and urine but not from the aqueous humor of the 2 febrile horses. The sera of all 4 challenged horses developed microscopic agglutination test antibody after challenge and remained relatively constant for 21 days. Titer to cross-reacting strains declined earlier than titer to the challenge strain. CONCLUSIONS: Clinical disease in experimentally infected horses can be mild or inapparent in Leptospira infected horses. Repeated serologic testing can allow recognition of the infecting serovar. In febrile horses, Leptospira can be isolated from blood while isolation from the urine can occur after fever has subsided.

Leptospirosis: An important infectious disease in North American horses.

North American horses are commonly exposed to Leptospira organisms. Leptospira Bratislava is the most common infecting serovar but this serovar has not been confirmed to cause clinical disease in North American horses. Leptospira Pomona type kennewicki is responsible for most of the clinical diseases (leptospirosis) in North American horses. Leptospirosis is most commonly associated with diseases of the placenta and fetus, the kidneys and the eyes in horses. In-utero infections in pregnant mares may result in abortion, neonatal illness or birth of an antibody positive healthy foal. Acute renal failure in younger horses and recurrent uveitis in adult horses are other well documented clinical syndromes of leptospirosis. Abortions, neonatal disease and acute renal failure are caused by a subacute infection, while horses with Leptospira associated recurrent uveitis develop ocular disease months or years after the initial Leptospira infection. Diagnosis of Leptospirosis is made by a combination of antigen or antibody testing methods. Mares that abort following Leptospira infection have no additional clinical signs at the time of abortion but may shed the offending Leptospira spp. in the urine for several weeks. Antibiotic treatments are sometimes used in hopes of decreasing Leptospira shedding in infected horses or prophylactically in exposed pregnant mares but documentation of efficacy is lacking. Horses with Leptospira - associated acute renal failure can be successfully treated with antibiotics and supportive care. Recurrent uveitis is commonly associated with leptospirosis in North American horses and although horses may have chronic intraocular infection triggering an immune disease, systemic antimicrobial therapy has not been effective in eliminating the organism from the eye. An equine approved Leptospira Pomona type kennewicki vaccine is now available in North America.

A luminescence-based assay for evaluating bactericidal antibody to Borrelia burgdorferi in vaccinated horses' serum.

BACKGROUND: Current serological tests cannot discriminate between bactericidal Borrelia burgdorferi antibodies from others that are merely a response to Borrelia antigenic stimulation. OBJECTIVE: To develop a sensitive and convenient luminescence-based serum bactericidal assay (L-SBA) to identify serum borreliacidal activity. STUDY DESIGN: Prospective validation study and method comparison. METHODS: Serum samples were obtained either from archives of the Animal Health Diagnostic Center at Cornell University (N = 7) or from a vaccination trial (N = 238). Endogenous complement-inactivated serum sample was incubated with exogenic complement and B. burgdorferi ML23 pBBE22luc, which is able to process luciferin with luciferase and produce luminescence in viable Borrelia. After incubation, a light signal can be detected by using a luminometer to calculate the borreliacidal antibody titre. RESULTS: Components of the reaction mixture including spirochetes and complement from various sources and concentrations were tested to identify a reliable recipe for our complement-mediated L-SBA. We also applied this L-SBA on measuring bactericidal antibody activities and calculated the half inhibitory concentration (IC50 ) of serum samples from clinical collections. Furthermore, we analysed the L-SBA titres and anti-outer surface protein A (OspA) antibody levels from vaccinated horses using the multiplex assays and found that there is a relationship between results generated using these two different assays. The increases of L-SBA titres correlated with increases of anti-OspA antibody titre in sera (r = 0.423). MAIN LIMITATIONS: Immunoreactivity of commercial complement may differ from different batches. Clinical protection of borreliacidal antibody levels has not been determined. CONCLUSIONS: The L-SBA provided a sensitive and easy-operating platform for the evaluation of bactericidal antibody to B. burgdorferi, and we anticipated L-SBA would function well as an evaluation tool of vaccine efficiency in the future.

Search for Borrelia burgdorferi in kidneys of dogs with suspected "Lyme nephritis".

BACKGROUND: "Lyme nephritis" is a poorly characterized condition associated with proteinuria and often fatal renal failure in dogs with serological evidence of infection with Borrelia burgdorferi. OBJECTIVE: The aim of this study was to determine if intact B. burgdorferi organisms were present in the kidneys of serologically Lyme-positive dogs with histopathologic features of Lyme nephritis. ANIMALS: Twenty-six affected and 10 control dogs were identified over an 8-year period (1996-2004) in databases at Cornell University's College of Veterinary Medicine. Case inclusion required serologic evidence of natural exposure to B. burgdorferi and availability of renal tissue (frozen or paraffin embedded) exhibiting pathology consistent with Lyme nephritis. METHODS: Renal tissue samples were assessed using modified Steiner (silver) (MS) staining, immunohistochemistry (IHC), polymerase chain reaction (PCR) using 4 primer sets (eubacterial, B. burgdorferi, Bartonella, and canine genomic DNA), and fluorescence in situ hybridization (FISH) using a 5'-cy3-eubacterial probe for 16S rRNA. RESULTS: MS stain was positive in 1 case; IHC was negative in all cases. None of the B. burgdorferi or Bartonella PCR reactions was positive. Two of the B. burgdorferi FISH analyses were positive. CONCLUSIONS AND CLINICAL IMPORTANCE: Minimal evidence of the presence of intact B. burgdorferi or any other bacterial organism was found in the renal tissue of dogs with suspected Lyme nephritis. Direct renal invasion by B. burgdorferi organisms does not appear to be responsible for this syndrome.

DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.

Potent 2'-amino-, and 2'-fluoro-2'-deoxyribonucleotide RNA inhibitors of keratinocyte growth factor.

Reiterative in vitro selection-amplification from random oligonucleotide libraries allows the identification of molecules with specific functions such as binding to specific proteins. The therapeutic usefulness of such molecules depends on their high affinity and nuclease resistance. Libraries of RNA molecules containing 2'amino-(2'NH2)- or 2'fluoro-(2'F)-2'-deoxypyrimidines could yield ligands with similar nuclease resistance but not necessarily with similar affinities. This is because the intramolecular helices containing 2'NH2 have lower melting temperatures (Tm) compared with helices containing 2'F, giving them thermodynamically less stable structures and possibly weaker affinities. We tested these ideas by isolating high-affinity ligands to human keratinocyte growth factor from libraries containing modified RNA molecules with either 2'NH2 or 2'F pyrimidines. We demonstrated that 2'F RNA ligands have affinities (Kd approximately 0.3-3 pM) and bioactivities (Ki approximately 34 pM) superior to 2'NH2 ligands (Kd approximately 400 pM and Ki approximately 10 nM). In addition, 2'F ligands have extreme thermo-stabilities (Tm approximately 78 degrees C in low salt, and specificities).

Assay for L-pipecolate oxidase activity in human liver: detection of enzyme deficiency in hyperpipecolic acidaemia.

A direct assay method is described for L-pipecolate oxidase. The assay uses NaHSO3 to trap the L-alpha-amino [3H]adipate delta-semialdehyde (AAS) formed as a direct reaction product of L-pipecolate oxidase from L-[3H]pipecolic acid. The adduct so formed was separated from the substrate on Dowex 50 (H+) column. The product was identified as [3H]AAS by amino acid analysis after breaking down the adduct by boiling under acidic conditions. The assay is simpler and more specific than fluorometric methods; it is also more sensitive, requiring at most 16 micrograms of liver peroxisome-enriched protein per assay. We have used this assay procedure to detect L-pipecolate oxidase in skin fibroblasts obtained from a control subject and from patients of hyperpipecolic acidaemia and Zellweger syndrome and found that this enzyme activity is present in the control, but absent or decreased in the patients with the peroxisomal disorders.

Uptake and metabolism of delta 1-piperidine-2-carboxylic acid by synaptosomes from rat cerebral cortex.

delta 1-Piperidine-2-carboxylic acid (P2C), an intermediate of the L-lysine metabolic pathway in the brain, was studied for its uptake and metabolism in the synaptosome of the rat cerebral cortex. The results of this study showed that the uptake of P2C into the synaptosome was NA+- and temperature-dependent with a two-tier transport kinetic (Km = 2.6 and 0.7 microM; Vmax = 1.6 and 0.73 pmol/min/mg). P2C uptake was only moderately inhibited (approximately 20%) by L-lysine and its metabolites, L-pipecolic acid and L-alpha-aminoadipic acid at up to 100 microM, and the putative amino acid neurotransmitters, gamma-aminobutyric acid, L-glutamic acid and L-aspartic acid (25-31%) at 5-500 microM. The synaptosomal preparation only has a very low activity for metabolizing P2C to its product L-pipecolic acid. The metabolic activity for P2C was mainly contained in the 27,000 x g supernatant S2 fraction. Since P2C is the precursor of the putative neuromodulator L-pipecolic acid, the understanding of its uptake and metabolic characteristics in the brain should be of significance.

L-pipecolic acid metabolism in human liver: detection of L-pipecolate oxidase and identification of its reaction product.

L-Pipecolate oxidase, an enzyme that oxidizes L-pipecolic acid in the human liver has been demonstrated in the peroxisomal preparation. This enzyme oxidizes L-pipecolic acid with concomitant production of H2O2 in the peroxisome of the normal human liver. The immediate product of L-pipecolic acid oxidation has been identified as L-alpha-aminoadipate delta-semialdehyde. This reaction product was directly, and also after conversion to pipecolic acid by NaBH4 reduction, characterized by use of an amino acid analyzer and thin-layer chromatography. The pit fall of an indirect assay of L-pipecolate oxidase by means of the assay of alpha-aminoadipic acid formation was discussed.