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PubMed1996

Equine coronary hemodynamics during brief coronary occlusions at three levels of collateral function.

Adult-grade ponies were surgically instrumented with a Doppler flow probe and pneumatic cuff occluder on the left anterior descending coronary artery (LAD), sonomicrometry crystals and intraventricular micromanometer in the left ventricle, and catheters in the left atrium, anterior interventricular vein, and, in some animals, the LAD. Conscious-animal studies were begun 2 wk after surgery. Measured variables included regional left ventricular systolic function, end-diastolic wall thickness, oxygen extraction, lactate extraction, and hydrogen ion release. Changes in collateral perfusion were deduced from changes in these variables. Serial data were obtained during a 3-min LAD occlusion before stimulation of collateral function by the intermittent coronary occlusion method and during a 10-min LAD occlusion after 14 +/- 2 and 27 +/- 2 days of stimulation. Hemodynamic interpretation of data was based on a model of the equine coronary circulation consisting of collateral and arteriolar resistances in series. It was concluded that 1) chronic stimulation of collateral function leads to the emergence of a time-dependent reduction in total collateral resistance during acute coronary occlusion; 2) with enhancement of collateral function, the major resistance controlling collateral blood flow shifts from the collateral circulation to the recipient vessel arterioles; and 3) at a certain level of enhanced collateral function, coronary occlusion results in a triphasic blood flow response in collateral-dependent myocardium consisting of early hypoperfusion, transient hyperperfusion, and late autoregulated perfusion. This study demonstrates that chronic stimulation of collateral function is accompanied by specific alterations in coronary hemodynamics during acute coronary occlusion that hasten the recovery of ischemic myocardium.

Animals↗
PubMed2005

Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene.

PURPOSE: Pigmented paravenous chorioretinal atrophy (PPCRA) is an unusual retinal degeneration characterized by accumulation of pigmentation along retinal veins. The purpose of this study was to describe the phenotype of a family with PPCRA, determine the mode of inheritance, and identify the causal mutation. METHODS: Ophthalmic examination was performed on seven family members and serially detailed in the proband over a 3-year period. Blood samples were collected and DNA extracted. All 12 coding exons and the 5' promoter region of the crumbs homologue 1 (CRB1) gene were PCR amplified and DNA sequenced. In silico homology modeling was performed on the mutated protein domain. RESULTS: Subtle symmetrical chorioretinal atrophy in the inferior quadrant was the earliest clinical sign detectable within this family. Paravenous pigmentation occurred initially in the far periphery, progressing centrally, with atrophy later becoming more widespread, involving the nasal, then the temporal, and finally the upper quadrant. A novel, dominant Val162Met mutation within the fourth EGF-like domain of CRB1 cosegregates with the PPCRA phenotype. It is thought to affect domain structure, because codon 162 is involved in hydrogen bonding between the antiparallel beta-strands of the major beta-sheet, causing sufficient perturbation of the backbone that the domain-stabilizing hydrogen bond does not form or is weakened. CONCLUSIONS: PPCRA was dominantly inherited in this family, but exhibited variable expressivity. Males are more likely to exhibit a severe phenotype, whereas females may remain virtually asymptomatic even in later years. The PPCRA phenotype is associated with a Val162Met mutation in CRB1 which is likely to affect the structure of the CRB1 protein.

Adult↗
PubMed1992

Immunoreactive somatostatin in the rat ovary.

Immunoreactive SRIH was detected in the rat ovary (15.6 pg/mg wet weight, 520 pg/mg protein) and was localized to the granulosa cells (168 +/- 6 pg/10(6) cells). Serial dilution studies showed parallelism of the inhibition curve for synthetic SRIH-14 and those of extracts of whole ovary and media conditioned by granulosa cells. The quantity of immunoreactive SRIH released into granulosa cell conditioned media decreased with time, while the intracellular content remained relatively constant. Gel chromatography showed peaks of immunoreactivity co-eluting with SRIH-14 (38%), SRIH-28 (31%) and a high molecular weight component. The addition of synthetic SRIH-14 stimulated meiotic maturation in cumulus-enclosed rat oocytes, with dose dependency being observed at SRIH-14 concentrations between 1 and 1000 pmol/l. No evidence of pre-pro-SRIH gene expression could be demonstrated in either rat ovary or testis using both Northern analysis and reverse transcriptase/polymerase chain reaction amplification of polyadenylated RNA. SRIH may be produced in the ovary during a specific stage of ontogeny or by an alternative gene. It is also possible that SRIH is actively taken up and stored by granulosa cells without being produced locally.

Animals↗
PubMed1999

Modulation of androgen metabolism by estradiol-17beta and progesterone, alone and in combination, in human gingival fibroblasts in culture.

BACKGROUND: Androgens, particularly 5alpha-dihydrotestosterone (DHT), have anabolic effects on connective tissues and bone with implications on periodontal healing. This can be enhanced by estradiol-17beta (E-17beta), in synergy with androgen action. The effects of progesterone (P) contribute to plaque-induced inflammatory changes. The aim of this investigation was to study the modulation of androgen metabolism by E-17beta and P, alone and in combination. METHODS: Human gingival fibroblasts were established in monolayer culture and duplicate incubations were performed in Eagle's MEM for 24 hours with either 14C-testosterone (14C-T) or 14C-4-androstenedione (14C-4-A) as substrate and serial concentrations of E-17beta, P and E-17beta + P. The medium was solvent extracted for steroid metabolites, analyzed, and quantified using a radioisotope scanner. The androgen substrates were converted mainly to DHT and 4-androstenedione/testosterone from 14C-T and 14C-4-A respectively. RESULTS: At concentrations of 0.1 and 0.5 microg/ml, E-17beta stimulated DHT synthesis from 14C-T by 18% and 12%, respectively, decreasing to control values at 0.1 microg/ml. While the effect of similar concentrations of P on the same substrates was inhibitory by 18, 70, and 82% (n = 4; P <0.01). E-17beta + P showed a 12% increase in DHT synthesis over controls at 0.1 microg/ml, similar to that of E-17beta alone, despite the inhibitory effects of P (n = 4; P <0.01) with 12% and 77% decreases at 0.5 and 1 microg/ml (n = 4; P <0.01). The inhibitory effect of P on DHT synthesis was less apparent when E-17beta was present in combination. The formation of 4-androstenedione from 14C-T was stimulated by E-17beta (12.5%), inhibited by P (50%) and showed an intermediate response with E + P (33% stimulation). At the concentrations used, E-17beta stimulated DHT synthesis from 14C-4-A by 3.6-, 3- and 2.6-fold. P also stimulated this conversion from the same substrate by 16%, 2-fold, and 1.6-fold increases, partly due to the low yields at baseline. The combination of E + P stimulated the synthesis of DHT from 14C-4-A by 4-fold at 0.1 and 0.5 microg/ml and a 2.3-fold increase at 1 microg/ml. The formation of T from 14C-4-A was stimulated by E-17beta (50%) and inhibited by P (40%), with 93% stimulation by E + P at 0.1 microg/ml. CONCLUSIONS: The modulatory effects of estradiol-17beta and progesterone on androgen metabolism may influence disease presentation and the progress of healing responses in the inflamed periodontium.

Adult↗
PubMed2003

[Cloning of human uracil N-glycosylase and its detection in cancer tissues by quantitative RT-PCR].

The uracil in DNA comes from either the misincorporation of dUTP in place of dTTP or deamination of cytosine. In the latter case, it can result in a GC to AT transition mutation if the uracil is not removed before DNA replication. Base excision repair (BER) is a major pathway for removing DNA lesions arising from endogenous processes as well as those induced by exposure to exogenous chemicals or irradiation. BER is initiated by DNA glycosylases that excise aberrant bases from DNA by cleavage of the N-glycosidic bond linking to the base of its deoxyribose sugar. Uracil N-glycosylase (UNG) is the enzyme responsible for the first step in the BER pathway that specifically removes uracil from DNA. The UNG gene undergoes both temporal and spatial regulation mainly at the level of transcription. Normally cancer cells undergo over-proliferation and up-regulate their UNG during tumorigenesis. In this study we examine the correlation between UNG level and carcinogenesis, and explore the possibility of using UNG as a marker for cancer diagnosis. Human UNG gene was amplified from the total RNA of the human choriocarcinoma cell line, JEG-3, by RT-PCR. After purification, the 942bp full-length UNG cDNA coding sequence was digested with EcoR I and Sal I, and cloned into the digested pET-21 to construct a recombinant vector, pUNG. The UNG protein was expressed under the control of T7 promoter in E. coli BL21 (DE3) cells induced with IPTG. After ultrasonic treatment, the cell lysate and precipitate were analyzed by SDS-PAGE and a 39kD band was detected. The plasmid was serially diluted at appropriate concentrations and employed as standards in the subsequent quantification. Total RNAs were extracted from 18 pairs of clinical samples, each pair contains a sample of esophageal squamous cell carcinoma (ESCC) tissue and its surrounding normal esophageal epithelia. The copy numbers of UNG mRNA in these RNA samples were determined by real-time quantitative RT-PCR using a Lightcycler (Roche). UNG was present in 13 cases of ESCC (13/18, n = 18) but absent in all of the normal tissues. The results indicated that there was a correlation between high level of UNG expression and the carcinogenesis of ESCC.

Carcinoma, Squamous Cell↗
PubMed1990

Pulp calcifications in primary teeth.

One hundred and twenty primary maxillary and mandibular extracted teeth were collected to study the occurrence and nature of pulp calcifications. The teeth were serially sectioned, stained with hematoxylin and eosin, examined by light microscope. Pulp calcifications were observed in 31 (25.8%) teeth. Two types of pulp calcifications were noticed i.e., diffuse calcifications and free/attached type denticles. The occurrence of pulp calcification appears identical in all teeth except in the first primary molar which is statistically not significant (P greater than 0.05). The low prevalence of pulp calcifications in the primary teeth support the view that pulp calcification increases as the age advances.

Dental Pulp Calcification↗
PubMed1989

Relationship of cytosolic estrogen and progesterone receptor content and the growth fraction in human mammary carcinomas.

In this study of breast cancer specimens, the relationship between cytosolic estrogen (ER) and progesterone receptor (PR) content to the size of the respective growth fraction (GF) (expressed as percentage of proliferating tumor cells) was investigated. We applied the recently developed ligand-binding assay for extracts of frozen sections and Ki-67 immunocytochemistry for the assessment of the GF to adjoining serial sections of a single tissue block. If the receptor content is plotted against the percentage of Ki-67 labeled cells, an inverse relationship between receptor content and proliferation becomes obvious, meaning that a high receptor content is associated with a small GF and vice versa. If tumor specimens are grouped according to the evaluated receptor status, the mean percentage of Ki-67-positive cells is 12% for ER-positive/PR-positive (ER+/PR+), 26% for ER-positive/PR-negative (ER+/PR-), 55% for ER-negative/PR-positive (ER-/PR+), and 57% for ER-negative/PR-negative (ER-/PR-) specimens. A significant population of tumors exists, however, which exhibit a high receptor content and a high GF. The percentages of ER+/PR+ samples with a high proliferation index are 16 and 26% if the total ER+ population is considered.

Biomarkers, Tumor↗
PubMed2003

Diversity of bacteria and Archaea in sulphate-reducing enrichment cultures inoculated from serial dilution of Zostera noltii rhizosphere samples.

We have analysed the diversity of culturable sulphate-reducing bacteria (SRB) in Zostera noltii colonized sediments from Bassin d'Arcachon (France). Four organic substrates have been tested as well as the combination of H2 and CO2 to select for lithotrophic SRB. All energy sources were supplied in parallel cultures that were amended with yeast extract plus NH4+ and prepared without a source of combined nitrogen, the latter to select for diazotrophic SRB. The 10 different enrichment media were inoculated from serial dilution of rhizosphere samples. The highest dilution cultures yielding positive growth (i.e. 10-7) were studied by molecular techniques (16S rDNA clone libraries, RISA and ARDRA). Lactate as a single organic substrate in combination with a source of combined nitrogen resulted in selection of members of the Desulfovibrionaceae. Surprisingly, when lactate was added without a source of combined nitrogen, Desulfobacteriaceae were selected. A strong influence of the presence or absence of combined nitrogen was also observed for the substrates sucrose and fructose. Whereas the liquid culture growing on sucrose and NH4+ systematically yielded 16S rDNA clones related to an environmental unidentified green sulphur bacterium (OPS185), on plates we were able to isolate a SRB related to Desulfovibrio dechloracetivorans, which likely represents a non-described species. Under diazotrophic conditions, sucrose selected for SRB clones related to the cluster formed by Desulfovibrio zosterae, Desulfovibrio salexigens and Desulfovibrio bastinii. The corresponding isolate obtained on plates showed only low sequence similarity with this closest neighbour (93.8%), and we suggest that it also represents a non-described species. Surprisingly, a 16S rDNA sequence corresponding to an archaeon, i.e. a non-extremophile Crenoarchaeota, was retrieved from several of the SRB enrichment cultures even after subsequent transfers.

Acetates↗
PubMed2000

Cocaine metabolism in hyperthermic patients with excited delirium.

The half-life of cocaine in clinical experiments has been reported to range from 60 to 90 min. It has been previously suggested that elevated temperature may accelerate the metabolism of cocaine. However, there is no clinical data to indicate the presence of hyperthermia like that seen in excited delirium alters the half-life of cocaine. We report the results of half-life determinations from serial cocaine concentrations in two patients with excited delirium. Both patients presented to the emergency department with classic findings of excited delirium that included hyperthermia, agitation, and cardiovascular aberrations. One patient died despite aggressive therapeutic intervention. Cocaine and metabolite concentrations were determined by an extractive alkylation mass spectrometry procedure. Presenting cocaine concentrations in patient 1 and patient 2 were 0.387 and 0.266 mg/L respectively. Results from pharmacokinetic modeling of the serial concentrations show that the half-life of cocaine was not significantly accelerated, despite the presence of hyperthermia. Data from these two cases provide further evidence that catastrophic reactions to cocaine are independent of amount or route of administration, and that the metabolism of cocaine, at least in these patients, was not altered by hyperthermia.

Journal Article↗
PubMed2006

Characterization of Fe(III)-reducing enrichment cultures and isolation of Fe(III)-reducing bacteria from the Savannah River site, South Carolina.

The Savannah River site, South Carolina (SRS), has been subjected to heavy metal and radionuclide contamination. Dissimilatory Fe(III)-reducing bacteria, by reducing insoluble Fe(III) to soluble Fe(II), may enhance contaminant mobility through subsurface environments. In order to investigate populations of the indigenous iron-reducing microbes from the SRS, duplicate enrichment cultures were initiated using a 10% inoculum of 7 sediment/soil samples, and serial dilutions were made into Wolfe's minimal salts media amended with 50 mM Fe(III) floc, 10 mM acetate and 0.01% yeast extract. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to generate fingerprints of the cultures and track changes in the microbial communities through the dilutions. Cluster analysis determined the relatedness of individual fingerprints. Initial enrichment cultures exhibited complex fingerprints consisting of many individual T-RF peaks, and demonstrated low similarity between sites. After four serial dilutions the fingerprints were less complex and clustered at higher similarities. Several individual T-RF peaks became dominant in a majority of the fingerprints. Cloning and sequence analysis revealed the presence of microbes closely related to Clostridium and Bacillus species and to known iron reducers such as Geobacter species and Pantoea agglomerans. Several Fe(III)-reducing isolates related to Aeromonas, Bacillus and Clostridium species were obtained.

Aeromonas↗
PubMed1991

Assay for the major dog allergen, Can f I: investigation of house dust samples and commercial dog extracts.

Monospecific rabbit antibodies were used to develop a sensitive two-site enzyme immunoassay to measure a major dog hair and dander allergen, Can f I. This Can f I assay demonstrated no reaction with 17 heterologous allergen sources, including dog albumin, cat, guinea pig, and horse. Analysis of serial dilutions of purified Can f I and the international standard for dog was parallel. The assay was considered specific for Can f I with a lower limit of detection at 0.03 micrograms/ml. Total imprecision was from 2% to 6%. Commercial dog extracts for specific immunotherapy contained from 0.7 to 290 micrograms of Can f I per milliliter. The assay was used to measure Can f I in 136 house dust samples collected from 103 homes across the United States. Concentration of the dog allergen was expressed as micrograms of Can f I per gram of dust. Prevalence of Can f I in the dust samples ranged from less than 0.3 to 10,000 micrograms/gm. Serial dilutions of samples containing Can f I were parallel to the standard. The median Can f I value for homes with a dog in residence was 120 micrograms/gm, and for homes with no dog, 3 micrograms/gm. With few exceptions, homes with no dog in residence had less than 10 micrograms/gm. This Can f I assay will provide useful information for assessing commercial extracts as well as monitoring dog-allergen exposure and allergen-control methods.

Allergens↗
PubMed2003

An assessment of two Carpobrotus species extracts as potential antimicrobial agents.

For centuries, indigenous people in South Africa have used a variety of medicinal herbs to treat chronic infections. This investigation focused on two Carpobrotus species belonging to the family, Aizoaceae, in an attempt to assess their antimicrobial potential. Extracts of varying polarities of the plants were prepared and tested against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Mycobacterium smegmatis. For the disc diffusion method, Ciprofloxacin (40 microg/disc) served as positive control for S. aureus, P. aeruginosa and M. smegmatis, whereas amphotericin B (25 microg/disc) was the control for C. albicans. A sample concentration of 10 mg/ml was used. Minimum inhibitory concentrations (MIC) were determined by two-fold serial dilution. Phytochemical analysis was completed to test for the presence of flavonoids, hydrolysable tannins, phytosterols and aromatic acids. The ethyl acetate extracts (21 microl of 95 mg/ml) were used for bio-autography, together with TLC analyses. Carpobrotus muirii and Carpobrotus quadrifidus showed antimicrobial activity against S. aureus and M. smegmatis in the disc diffusion method and inhibition against S. aureus and M. smegmatis was observed by clear zones on the TLC plate. This investigation confirms that extracts of these Carpobrotus species that are used as indigenous medicines, exhibit anti-bacterial activity. This scientific information can serve as an important platform for the development of inexpensive, safe and effective natural anti-infective medicines.

Anti-Bacterial Agents↗
PubMed1993

Susceptibility of human liver cell cultures to hepatitis C virus infection.

To develop a cell culture system susceptible to infection by hepatitis C virus (HCV), human fetal hepatocytes, grown in serum-free medium, were inoculated with serum samples from two HCV-infected patients. Viral RNA sequences were detected by polymerase chain reaction, using primers specific for the 5' noncoding region of HCV, in extracts prepared from the hepatocyte cultures as early as 5 days after inoculation. Virus was also released from the infected cells into the medium. The HCV strains could be serially passaged three times into fresh liver cell cultures using intracellular virus as inoculum. Evidence that HCV replication really took place in primary human fetal hepatocytes was also obtained by detection of minus-strand viral RNA (replication intermediate) in cell extracts and of viral antigens in the infected cells.

Base Sequence↗
PubMed2000

Expression of transforming growth factor-alpha mRNA in livers of patients with chronic viral hepatitis and hepatocellular carcinoma.

BACKGROUND: Transforming growth factor-alpha (TGFalpha) is an important autocrine growth factor of hepatocytes. The authors evaluated the roles of TGFalpha in chronic viral hepatitis (CVH) and hepatocellular carcinoma (HCC). METHODS: The authors measured the amounts of TGFalpha mRNA in liver tissues from 18 patients with HCC, 31 patients with CVH, and 7 normal controls. " Hot-start" reverse transcription-polymerase chain reaction (RT-PCR) using oligo-dT and specific primers detected TGFalpha mRNA in total cellular RNA extracted from liver tissues. The levels of TGFalpha mRNA were determined by the end point titers of serial, two-fold dilutions of cDNA. The amounts of hepatitis B virus RNA (HBV-RNA) in livers of patients with chronic hepatitis B also were measured by Northern blot hybridization. RESULTS: TGFalpha mRNA levels were extremely higher in patients with HCC compared with patients with CVH and normal controls, and the levels in patients with CVH also were elevated compared with normal controls. The levels of TGFalpha mRNA were overexpressed in the underlying livers of patients with HCC compared with patients with CVH, although they were lower than those found in HCC tissues. The levels of TGFalpha mRNA were higher in samples from patients with chronic hepatitis B than in samples from patients with chronic hepatitis C. The levels of TGFalpha mRNA were not correlated with serum alanine aminotransferase or HBV-RNA levels in liver tissues in patients with chronic hepatitis B. However, the expression of TGFalpha mRNA tended to be higher in the livers of patients with raised serum alpha-fetoprotein levels. CONCLUSIONS: The overexpression of TGFalpha mRNA in the liver seems to be associated with the regeneration of hepatocytes rather than hepatic necrosis or viral replication. Also, it may be related closely to the development or progression of HCC, especially in the livers of patients with chronic hepatitis B.

Adult↗
PubMed1998

Laparoscopic splenectomy in beta-thalassemia major patients. Advantages and disadvantages.

BACKGROUND: Splenectomy is indicated in patients with thalassemia major when they develop hypersplenism with subsequent need for increased transfusions. Extreme splenomegaly is considered a restrictive factor for laparoscopic splenectomy in these patients. METHODS: Laparoscopic splenectomy was undertaken in 12 beta-thalassemia major patients with massive splenomegaly. The devascularization of the organ was performed with serial ligations of the splenic vessels starting from the lower pole of the organ. The spleen was extracted from the abdominal cavity through a 5-cm incision in the left iliac fossa, which incorporated two port sites. RESULTS: The procedure was concluded laparoscopically in 10 cases, while two patients were converted due to difficulty in controlling bleeding from branches of the splenic vein. The patients tolerated the procedure well and had a postoperative hospital stay of 3-6 days. CONCLUSIONS: From our limited initial experience it seems that laparoscopic splenectomy in the difficult setting of thalassemia major patients is feasible, but extreme care is required in order to avoid hemorrhagic complications.

Adolescent↗
PubMed1989

Anti-histone autoantibodies in ddY mice, an animal model for spontaneous IgA nephritis.

The ddY mice are known to develop spontaneous glomerulonephritis resembling human IgA nephritis after 40 weeks of age. A sharp rise of circulating polyclonal IgG and IgA is also observed at this stage. Since these overproduced immunoglobulins seem to be related to the development of murine glomerulopathy, antigen-antibody interactions between renal tissue proteins and serum immunoglobulins were analyzed by Western blotting in ddY mice before and after 40 weeks of age. Serum IgG at 50 weeks reacted with an 18-kDa renal tissue protein which was identified as histone H3, as well as with histone H1. Renal histones were extracted along with IgG from the murine kidney at 50 weeks in a high salt soluble fraction. Serial studies of anti-histone antibodies by enzyme-linked immunosorbent assay showed that IgG class antibodies markedly increased after 40 weeks of age. IgA class antibodies mildly increased after 56 weeks of age. Anti-DNA antibodies were not detected. These results demonstrate that ddY mice also develop mainly IgG class and partly IgA class anti-histone autoantibody after 40 weeks of age, and that histone-anti-histone complexes may contribute to the development of murine glomerulopathy. Although anti-histone antibodies have been reported in lupus mice, ddY mice differ from these mice in that no anti-DNA antibodies develop.

Animals↗
PubMed2001

Nucleic-acid sequence based amplification in the rapid diagnosis of rabies.

Current serological tests do not reliably diagnose rabies. We describe a technique based on amplification of nucleic-acid sequences to detect rabies-specific RNA in the saliva and cerebrospinal fluid (CSF) of four living patients with rabies. Rabies RNA could be detected in either saliva or CSF, or both, in all patients and as early as day 2 after onset of symptoms. Both saliva and CSF should be serially tested because not every sample can be expected to be positive. The whole process, including automated extraction, isothermal amplification, and detection can be done within 4 h.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Serial measurement of cross-sectional area in peripheral vein grafts using three-dimensional ultrasound.

Frequent surveillance of bypass grafts placed in the lower limbs can provide early detection of stenoses. A three-dimensional (3-D) ultrasound (US) imaging system has been used to produce serial surface reconstructions of regions of interest in vein grafts in the lower extremities. Using anatomical reference points, data sets from serial studies are registered in a common 3-D coordinate system. Cross-sectional area measurements are extracted from the surface reconstructions in planes normal to the vessel center axis. These measurements are compared at matched sites over time to track changes in the vessel configuration. The quantitative measurements are paired with surface displays of the vessels for a complete depiction of the changing geometry. Example studies from three patients are shown, for time periods up to 38 weeks. The cross-sectional area measurements highlight regions of remodeling and developing stenoses within the grafts.

Adult↗