Chromatin structure changes suggest a compensatory response to c-myc gene amplification in malignant fibrous histiocytoma. Changes in chromatin structure as determined from DNAse I hypersensitive site analysis are associated with c-myc amplification and increased transcript/protein levels in malignant fibrous histiocytoma (MFH) cell lines. A DNAse I hypersensitive site near the PO promoter region was observed in one MFH cell line (UR HCL 1), and in normal fibroblasts (HFF), but not in an MFH cell line with an amplified c-myc gene (P3C). A DNAse I hypersensitive site exclusive to P3C amplified c-myc was identified slightly 3' of exon one. No alterations in c-myc DNAse I hypersensitive site patterns were observed in HFF fibroblasts following serum release, when peak levels of c-myc transcript were induced. DNAse I hypersensitive site patterns associated with gene amplification may reflect a compensatory response by P3C cells to an abundance of c-myc transcript. Furthermore, elevated levels of protein in P3C cells provide additional evidence that amplified c-myc is an oncogene in MFHs.


Monensin inhibits the binding of 3H-flunitrazepam to and reveals the intracellular passage of GABAA/benzodiazepine receptor. Effects of monensin were examined on the intracellular processing of the GABAA/benzodiazepine receptor (GABAA/BZDR) in neuron cultures derived from embryonic chicken brain, using 3H-flunitrazepam as the probe for the benzodiazepine modulator site on the receptor. Incubation of cultures with 0.1 or 1 microM monensin for 3 h blocked the binding of 3H-flunitrazepam by about 18%. Loss of ligand binding was due to a reduction in the number of binding sites, with no significant changes in receptor affinity. The general cellular protein synthesis and glycosylation in the cells were inhibited by 26% and 56%, respectively, in the presence of 1 microM monensin, as detected by assaying the incorporation of 3H-leucine and 3H-galactose. In contrast, an increase was observed for mannose incorporation by the cultures in the presence of the drug. Moreover, the results from in situ trypsinization of the cultures following monensin treatment showed that monensin did not alter the distribution of intracellular and surface receptors. The data suggest that monensin induces the down-regulation of GABAA/BZDR by generating abnormal glycosylation of the receptor and interrupting its transport within the Golgi apparatus, as well as from the Golgi apparatus to the intracellular pool and cell membrane. The galactosylation of receptor proteins may be important for the maturation of the receptor.


Overexpression of the csA cell adhesion molecule under its own cAMP-regulated promoter impairs morphogenesis in Dictyostelium. The contact site A (csA) glycoprotein is a strictly developmentally regulated plasma membrane component responsible for the EDTA-stable (Ca(2+)-independent) form of intercellular adhesion in Dictyostelium discoideum. Using inverse polymerase chain reaction and a terminator vector we have isolated a 1.6 kb genomic fragment carrying a 1.1 kb upstream region of the csA gene. This fragment had promoter activity in D. discoideum cells, giving rise to a 3'-truncated csA RNA that was regulated like the mRNA of the endogenous gene. Cyclic AMP pulses strongly enhanced transcription from the cloned csA promoter. These findings provide evidence that the cloned region of the csA gene comprises the complete promoter. It contains a G/C-rich octamer motif similar to other cAMP-regulated D. discoideum promoters. When the csA protein was strongly overexpressed under the developmental control of the csA promoter, morphogenesis was substantially altered. Aggregation was delayed, and secondary centres were formed along aggregation streams that led to fragmentation of the aggregates and multiple slug formation. At high cell density a substantial portion of aggregated cells was left behind on the substratum when slugs and fruiting bodies were built. The transformation vector was also employed to rescue a csA-negative mutant, HG1287, from its cell adhesion defect.


Association of cyclin-bound p34cdc2 with subcellular structures in xenopus eggs. Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM beta-glycerophosphate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2-cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdc2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.


Adhesion of 8701-BC breast cancer cells to type V collagen and 67 kDa receptor. Ductal infiltration carcinomas (d.i.c.) of the breast are potentially highly metastatic tumours, associated with drastic alterations of the architecture and molecular composition of the extracellular matrix at the tumour-host interface. 8701-BC, a recently characterized cell line, isolated from primary d.i.c., was used to study different aspects of tumor cell-substratum interactions. Since type V collagen deposition is augmented in d.i.c. we have examined the ability of 8701-BC cells to interact with this collagen species. We have found that cell binding to type V collagen was mediated by protein homologous to the 67 kDa laminin receptor (67-R). This conclusion is substantiated by the following observations: (a) a major band having an apparent molecular mass of 67 kDa and immunoreactive to the anti-67 R antibody was detectable by SDS-PAGE of the membrane proteins; (b) the antibody inhibited cellular adhesion to type V collagen in a dose-dependent way; (c) membrane proteins purified by affinity chromatography on type V collagen were immunoreactive to anti-67 R antibody, but not to anti-VLA1, VLA2 and VLA3 integrin antibodies. This receptor appears to have prominent carbohydrate-binding properties, since lactose competes with cell adhesion to type V collagen.


Maturation of cerebral oxidative metabolism in the cat: a cytochrome oxidase histochemistry study. The maturation of brain oxidative capacity was studied in kittens, using cytochrome oxidase histochemistry, at different ages throughout development. Optical densitometry values of reacted tissue were obtained for 50 different structures of the brain. In general, most structures reached adult levels of oxidative capacity by 30 days of age with some motor areas (e.g., cerebellum, red nucleus) exhibiting adult values as early as 7 days of age. Thereafter, some structures (e.g., basal ganglia, thalamus) exhibited levels of cytochrome oxidase activity that exceeded adult values for varying periods of time. These findings indicate regional heterogeneity in the maturation of cerebral oxidative capacity. Furthermore, these maturational patterns appear to correlate well with previous observations from anatomical, physiological and neurobehavioral studies.


Equivalence of a high-performance liquid chromatographic assay and a bioassay of azithromycin in human serum samples. Two sensitive methods for the determination of the azalide antibiotic azithromycin in human serum were compared. High-performance liquid chromatography (HPLC) and a microbiological assay were simultaneously applied to 768 serum samples obtained in a clinical study. There was excellent agreement between the azithromycin concentrations measured by HPLC and by the bioassay. The correlation coefficient for the two methods was r2 = 0.96. The precision and the sensitivity of the methods were found to be very similar.


Separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography. The separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography is presented. Total lipid extracts from Escherichia coli and human spermatozoa were loaded onto pre-packed aminopropyl-bonded silica gel columns and the lipids separated into four fractions. Non-polar lipids including cholesterol esters, triglycerides, diglycerides, monoglycerides and cholesterol, were eluted with 4 ml of isopropanol-chloroform (1:2, v/v) (fraction 1); free fatty acids were eluted with 4 ml of 2% acetic acid in diethyl ether (fraction 2); neutral polar lipids, including phosphophatidylethanolamine, phosphatidylcholine, sphingomyelin and neutral glycolipids, were eluted with 4 ml of methanol (fraction 3); and, finally, polar acidic lipids, including phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, seminolipid lipid A and acidic glycosphingolipids, were eluted with 4 ml of chloroform-methanol-0.8 M sodium acetate (60:30:4.5, v/V/V) (fraction 4). The recoveries for the different lipids ranged between 89 and 98% and the intra-assay variation, expressed as the standard deviation, was less than 5%.


High-performance liquid chromatographic determination of 2',3'-didehydro-3'-deoxythymidine, a new anti-human immunodeficiency virus agent, in human plasma and urine. Sensitive and selective high-performance liquid chromatographic techniques have been developed for the determination of 2'-3'-didehydro-3'-deoxythymidine, d4T (BMY-27857), in human plasma and urine. The methods had linear standard curves over the concentration ranges 0.025-25.0 and 0.5-100 micrograms/ml for the plasma and urine matrices, respectively. Both methods used solid-phase extraction for isolating d4T and the internal standard, thymidine oxetane, from the biological matrix. In addition, the analytical column, mobile phase, instrumentation and chromatographic conditions used for both methods were identical. The ultraviolet absorbance of the column effluent was monitored at 266 nm. Results of analysis of quality control samples indicated that the intra-assay precision values, as measured by percent relative standard deviation, were within 12 and 3%, and accuracy samples deviated less than 10 and 5% from nominal values for the plasma and urine assays, respectively.


Thermospray liquid chromatographic-mass spectrometric analysis of anti-AIDS nucleosides: quantification of 2',3'-dideoxycytidine in plasma samples. Thermospray liquid chromatography-mass spectrometry was investigated as a method for quantification of 2',3'-dideoxycytidine (DDC) from human plasma. A stable isotope analog of DDC ([15N2,2H2]DDC) was used as an internal standard. Selected ion monitoring of the protonated molecular ions for DDC and the internal standard was used to record mass chromatograms. The areas of the peaks in the mass chromatograms were used for quantification. The detection limit of DDC in this assay was 50 pg on-column. The calibration curve was linear over the desired range, 0.25-20 ng/ml. The major advantages of this assay over others are: no derivatization, high sensitivity, high specificity and short assay time.


Determination of a cyclic heptapeptide, a novel fibrinogen receptor antagonist, in human plasma by high-performance liquid chromatography with automated pre-column derivatization, column switching and fluorescence detection. A sensitive high-performance liquid chromatographic (HPLC) assay for the determination of the cyclic heptapeptide Ac-Cs-Asn-Dtc-Amf-Gly-Asp-Cys-OH (Dtc = beta,beta-dimethylthioproline, Amf = p-aminomethylphenylalanine) in human plasma has been developed. The key steps in the assay include: solid-phase extraction of the drug from plasma, chemical derivatization of the primary amino group with naphthalene-2,3-dicarboxyaldehyde in the presence of N-acetyl-D-penicillamine as a nucleophile to form a fluorescent benzo[f]isoindole derivative, and HPLC with column switching to provide the necessary chromatographic separation of the derivative from endogenous plasma components. The assay has been validated in the concentration range 1-10 ng/ml of plasma.


Determination of enantiomeric concentrations of a 2,5-diaryltetrahydrofuran (L-668,750), a platelet-activating factor receptor antagonist, in rat plasma using a chiral alpha 1-acid glycoprotein high-performance liquid chromatographic column. Racemic sulfonylated 2,5-diaryltetrahydrofuran [L-668,750, (+-)-trans-2-[3-methoxy-5-(2-hydroxy)ethylsulfonyl-4-n-propoxy]-p henyl-5-(3,4,5-trimethoxyphenyl)-tetrahydrofuran, I] is a potent, specific and orally active platelet-activating factor (PAF) receptor antagonist. Its (-)-(2S,5S) enantiomer [L-680,573, (S)-I] exhibited higher PAF antagonistic potency than the (+)-(2R,5R) enantiomer [L-680,574, (R)-I] in vitro and in animal models. For assay of drug concentrations in plasma of rats dosed intravenously or orally with tritium-labeled I, we have developed a high-performance liquid chromatographic (HPLC) method which directly resolved the two enantiomers. The column contained alpha 1-acid glycoprotein as the chiral stationary phase and was eluted with phosphate buffer, methanol and ethanol at neutral pH. The concentration of each enantiomer in the plasma was then determined by reverse isotope dilution assay. Results showed that the plasma clearance rate of the more potent (S)-I enantiomer was more than ten-fold faster than that of the (R)-I enantiomer; the enantioselective clearance resulted in nearly ten-fold higher concentrations of the latter in plasma at all time points regardless of the dosing route. This paper describes the HPLC chiral resolution method and its application in plasma analysis.


Gas chromatographic-mass spectrometric methods of analysis for detection of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in biological matrices. Gas chromatographic-mass spectrometric methods of analysis for the detection of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid, a major metabolite of delta 9-tetrahydrocannabinol, are reviewed. Emphasis is on analytical methodology including numerous derivatization techniques developed specifically for this analyte. The majority of procedures cited in the literature were developed to detect this metabolite in the blood and urine of man.


Determination of divalent trace metals in natural waters by preconcentration on N,N,N',N'-tetra(2-aminoethyl)ethylenediamine-silica followed by on-line ion chromatography. A method for the on-line preconcentration and chromatography of trace metals, e.g., Co, Ni, Cu, Zn, Cd and Pb, on N,N,N',N'-tetra(2-aminoethyl)ethylenediamine-bonded silica is described. The preconcentrated metals were desorbed with 0.13 M tartrate, which allows direct separation on a cation-exchange chromatographic column. The metals separated were detected by postcolumn reaction with 4-(2-pyridylazo)resorcinol and measuring the absorbances at 500 nm. Linear calibration graphs were obtained over the range 1.10(-8)-3.10(-6) M. The synthesis and characteristics of the chelated silica are described. The method was applied to the analysis of river and interstitial sediment waters.


High-performance affinity chromatography of DNA. II. Porosity effects. A DNA-silica, (dT)18-silica, was prepared and used in a study of the chromatography of the oligonucleotide, (dA)18, based upon base pairing. It was shown that hybridization efficiency did not depend upon flow-rates up to 2 ml/min for the small columns (22 x 2 mm) used. As increasing amounts of (dA)18 were loaded onto the columns, the columns were found to saturate at a well defined capacity that was always less than the amount that theoretically could have been bound. Maximum capacity was achieved whenever the loading temperature was at least 20-25 degrees C below the temperature at which the loaded oligonucleotide would elute. The effects of porosity on both coupling efficiency and capacity were measured and suggest that pore sizes in the 300-500 A range are most appropriate for this form of chromatography.


Drug matrix effect on the determination of residual solvents in bulk pharmaceuticals by wide-bore capillary gas chromatography. A wide-bore capillary gas chromatographic method has been developed to study the drug matrix effect on the determination of residual solvents in bulk pharmaceuticals. A selective method is achieved on a Restek wide-bore (0.53-mm i.d. x 30 m) open-tubular fused-silica column coated with a 5-micron film of 95% dimethyl-5% diphenyl polysiloxane protected by a phenyl-methyl siloxane deactivated, uncoated fused-silica guard column. Utilizing this method, several common process solvents in weakly acidic, weakly basic, and neutral drug matrices are evaluated by recovery and linearity studies to show whether or not a drug matrix effect exists in their determination.


Clothing prevents ultraviolet-B radiation-dependent photosynthesis of vitamin D3. Photoprotection of the skin is mainly a function of clothing, although the effectiveness of the latter against UV-B solar radiation (wavelengths 290-320 nm) has not been measured in vivo. Since UV-B mediates the cutaneous formation of vitamin D3, we examined the attenuation of that photosynthetic reaction by the commonly used fabrics cotton, wool, and polyester in black and white colors. Direct transmission of UV-B was attenuated the most by black wool (98.6% of incident irradiance) and the least by white cotton (47.7%). None of the fabrics allowed the photoproduction of previtamin D3 from 7-dehydrocholesterol irradiated in vitro with up to 40 min of simulated sunlight or the elevation of serum vitamin D3 after irradiation with approximately one minimal erythema dose (MED) of UV-B in volunteers wearing jogging garments made of these fabrics. Increasing the whole body irradiation dose to six MEDs still failed to produce a serum vitamin D3 response in garment-clad subjects. Regular (seasonal) street clothing also prevented an elevation of the vitamin D3 in response to UV-B radiation. We conclude that clothing prevents or significantly impairs the formation of vitamin D3 after photostimulation with up to six MEDs of UV-B.


Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. I. Experimental conditions to quantitate membrane-bound adenylyl cyclase activity. Although cyclic adenosine monophosphate (cAMP) is an important regulator of motility and metabolism in human spermatozoa, little is known on the cellular system responsible for its synthesis. Here, we investigated the experimental conditions directly to quantitate adenylyl cyclase (AC) activity synthesizing cAMP in human ejaculated spermatozoa and analysed the general properties of the enzyme. A 10,000 g membrane fraction was prepared from washed sperm cells homogenized by sonication. AC activity was monitored by the direct conversion of [alpha-32P]adenosine triphosphate (ATP) into [32P]cAMP. Using a nucleoside triphosphate regenerating system to ensure availability of ATP substrate, the human sperm AC showed a steady production of cAMP for at least 1 h. The assay was optimized for pH, buffer concentration, membrane protein and substrate concentration. Activity was dependent upon the presence of Mn2+ as a divalent cation and showed a pH optimum between 7.0 and 8.5. Optimal activity required 5 mM ATP, 1 mM ethylenediamine tetraacetic acid (EDTA) and 20-40 mM total MnCl2. Dependence on Mn2+ was not mandatory; Mg2+ at 5-40 mM also supported significant activity, but the activity was 4-6 times lower than that with Mn2+. Regardless of the presence of Mn2+ or Mg2+ as divalent cation in the assay, human sperm AC was insensitive to the regulatory ligands NaF, guanine nucleotide or forskolin. Insensitivity to these ligands supports the proposal that this enzyme system does not contain a stimulatory guanine nucleotide-binding regulatory protein and that its catalytic component is unique and different from that of somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. II. The role of calcium and bicarbonate ions on the activation of adenylyl cyclase. In this study, we have applied our previous data describing the experimental conditions necessary for expression of cyclic adenosine monophosphate (cAMP)-synthesizing adenylyl cyclase in human ejaculated spermatozoa, to investigate the direct effects of calcium (Ca2+) and bicarbonate (HCO3-) upon activation of the enzyme in vitro. We report that the effects of Ca2+ and HCO3- were significantly dependent on the status of the enzyme activity. Thus, at a near saturating (10 mM) concentration of MnCl2 giving high enzyme activity, addition of less than 10 mM HCO3- did not affect adenylyl cyclase activity and higher concentrations inhibited the enzyme, with 50 mM HCO3- reducing the activity by 33%. Also, addition of less than 20 mM CaCl2 alone or in combination with 10 mM HCO3- did not significantly change the enzyme activity. In great contrast, enzyme activation was highly responsive to Ca2+ and HCO3- when MnCl2 was present at a concentration giving submaximal enzyme activity. Thus, at 2 mM MnCl2, adenylyl cyclase was markedly increased by CaCl2 concentrations between 10 and 100 mM. The activation was further enhanced by increasing concentrations of HCO3-, with 50 mM HCO3- giving the highest activity at 50-100 mM CaCl2. Activation by CaCl2 was also observed in the absence of added MnCl2, being significantly greater than basal activity at 10 mM CaCl2 and maximal at 100 mM CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)


Polymerase chain reaction detection of human papillomavirus: quantitation may improve clinical utility. A case-control study compared detection by polymerase chain reaction (PCR) specific for human papillomavirus (HPV) type 16 with restriction enzyme analysis and Southern blot hybridization detection of HPV type 16. Cervicovaginal lavage samples from 64 women with histopathologic evidence of a cervical squamous intraepithelial lesion and 55 samples from cytologically healthy women were studied. Several methods of PCR product analysis, including radioactive and nonradioactive probing, were compared. The sensitivity of HPV detection by PCR when the amplified DNA fragment was visualized on a gel was equivalent to those of detection by restriction enzyme and Southern blot analyses. Hybridization of the PCR product with radioactively or nonradioactively labeled oligonucleotide probes increased the sensitivity of HPV detection by 100-fold. However, an increase in the sensitivity of the assay preferentially identified low levels of the virus in cytologically healthy women. Therefore, the value of HPV detection in identifying women with cervical neoplastic disease was greater, and the odds ratio for the presence of a cervical squamous intraepithelial lesion was higher when the less sensitive modalities were used. These results suggest that quantitation of HPV by PCR may maximize the clinical significance of a positive test result. Further studies will be needed to determine the optimal level of virus detection which has the highest positive predictive value of clinical disease.


Epidemiologic typing and delineation of genetic relatedness of methicillin-resistant Staphylococcus aureus by macrorestriction analysis of genomic DNA by using pulsed-field gel electrophoresis. To evaluate the usefulness of phenotypic and genotypic analyses for the epidemiologic typing of methicillin-resistant Staphylococcus aureus (MRSA), we characterized 64 epidemic MRSA isolates and 10 sporadic methicillin-susceptible S. aureus isolates from a university hospital and 18 MRSA isolates from hospitals in different geographical areas. Chromosomal DNA macrorestriction analysis with SstII was resolved by pulsed-field gel electrophoresis and compared with antibiotype analysis, phage type analysis, and standard genomic DNA restriction analysis with BglII. Indices of the discriminatory ability of these methods were 0.982, 0.959, 0.947, and 0.959, respectively. Macrorestriction patterns of 94% of MRSA isolates from patients, personnel, and the environment associated with a nosocomial outbreak were closely related (similarity coefficient, 85 to 100%). In contrast, methicillin-susceptible S. aureus isolates showed a marked diversity of macrorestriction patterns (median similarity, 41%). MRSA isolates from other geographical areas showed diverse macrorestriction patterns, with the exception of four isolates displaying identical or closely related patterns; these isolates were associated with concurrent outbreaks in four other Belgian hospitals. A concordance of genomic DNA macrorestriction typing with phenotypic methods was observed for 60 to 65% of MRSA isolates, and a concordance with standard DNA restriction analysis was found for 79 to 98% of these isolates. In conclusion, genomic DNA macrorestriction analysis was a useful complement to phenotypic methods for delineating epidemic isolates of MRSA, for identifying their nosocomial reservoirs, and for tracing their intra- and interhospital spread. The genetic relatedness of MRSA isolates, as estimated by this technique, appeared to correlate with their space-time clustering.


Use of a modified shell vial technique to quantitate cytomegalovirus viremia in a population of solid-organ transplant recipients. A quantitative modification of the shell vial assay was used to investigate cytomegalovirus viremia in solid-organ transplant recipients. The level of viremia detected in 109 of 407 specimens ranged from 0.02 to 28 infectious foci per 100,000 leukocytes. By using a Poisson model, a technique was developed to determine 95% confidence limits for the measured levels of viremia. These confidence limits were used to determine the level of viremia that could be excluded by culturing a given number of cells. Longitudinal assessment of two transplant recipients revealed different patterns of viremia and demonstrated that significant disease sometimes occurred with low-level viremia. On the basis of the results of the studies, culture of at least 4 x 10(6) leukocytes is recommended for the sensitive detection of cytomegalovirus viremia.


Characterization of full-length and polymerase chain reaction-derived partial-length Gottfried and OSU gene 4 probes for serotypic differentiation of porcine rotaviruses. To determine the VP4 (P type) specificity of porcine rotaviruses, full- and partial-length gene 4 probes were produced from cloned Gottfried and OSU porcine rotavirus genomic segment 4 cDNAs. The gene 4 segments from the prototype Gottfried (VP7 serotype 4) and OSU (VP7 serotype 5) porcine rotavirus strains were selected for study because of their distinct P types and the occurrence of rotaviruses with similar serotypes among swine. Partial-length gene 4 cDNAs were produced and amplified by the polymerase chain reaction (PCR) and encompassed portions of the variable region (nucleotides 211 to 612) of VP8 encoded by genomic segment 4. The hybridization stringency conditions necessary for optimal probe specificity and sensitivity were determined by dot or Northern (RNA) blot hybridizations against a diverse group of human and animal rotaviruses of heterologous group A serotypes and against representative group B and C porcine rotaviruses. The PCR-derived gene 4 probes were more specific than the full-length gene 4 probes but demonstrated equivalent sensitivity. The Gottfried PCR-derived probe hybridized with Gottfried, SB2, SB3, and SB5 G serotype 4 porcine rotaviruses. The OSU PCR-derived probe hybridized with OSU, EE, A580, and SB-1A porcine rotaviruses and equine H1 rotavirus. Results of the hybridization reactions of the PCR-derived gene 4 probes with selected porcine rotavirus strains agreed with previous serological or genetic analyses, indicating their suitability as diagnostic reagents.


Comparison of the Histopaque-1119 method with the Plasmagel method for separation of blood leukocytes for cytomegalovirus isolation. Histopaque-1119 (Sigma Chemical Co., St. Louis, Mo.) and Plasmagel (Cellular Products, Inc., Buffalo, N.Y.) were compared as density gradient separation reagents for the separation of polymorphonuclear leukocytes and mononuclear cells from blood from the isolation of cytomegalovirus (CMV). Of 200 peripheral blood specimens examined, CMV was recovered from 51 by both methods. The time of detection of immunofluorescent sites or a cytopathic effect associated with CMV was similar by each method. The Histopaque-1119 method was less time-consuming than the Plasmagel method since it did not require a precentrifugation step for the settling of erythrocytes. The use of Histopaque-1119 will permit an effective alternative single-step method for the separation of blood leukocytes for the isolation of CMV.


Antibody responses to synthetic peptides from cytomegalovirus phosphoprotein 150. We have identified antigenic regions within phosphoprotein 150 of human cytomegalovirus (CMV pp150) to which seroreactivity appears in patients with active CMV infection or persists in seropositive persons. A range of 8.3 to 61.6% of healthy CMV-seropositive blood donors were immunoglobulin G positive for single peptides, while 91.6% reacted to a mixture of four peptides. All convalescent-phase serum samples from 26 patients with active CMV infection reacted with either of two peptides encompassing amino acids (aa) 594 to 623 and aa 614 to 643. Patients with a primary CMV infection had patterns of reactivity to single peptides different from those of patients with reactivated CMV infection. The immunoglobulin M antibodies reacted preferentially with the peptides encompassing aa 594 to 663 of CMV pp150.


Comparison of indirect immunofluorescence and membrane fluorescence assays for the differentiation of antibodies to human immunodeficiency virus types 1 and 2. Serum samples from 20 human immunodeficiency virus type 1 (HIV-1)- and 30 HIV-2-infected and 7 dually infected individuals were reacted by using the indirect immunofluorescence assay (IFA) and membrane fluorescence assay in order to determine whether these methods were useful for typing HIV-1 and HIV-2 antibodies. Although 41 of 50 (82%) of the HIV-1- and HIV-2-positive specimens cross-reacted to some extent with the heterologous antigen in the IFA, the antigen with the higher titer correlated completely with the infecting type. The IFA could not distinguish single from dual infections, however. In contrast, only 4 of the 50 (8%) serum samples cross-reacted in the membrane fluorescence test. All seven of the specimens from patients with mixed infections reacted with both antigens. The membrane fluorescence test appears to be reliable for serodifferentiation of HIV-1 and HIV-2 infections and may be useful for laboratories with low-volume typing requirements.


Effect of interstrain variation on diagnostic DNA amplification of the cytomegalovirus major immediate-early gene region. The immediate-early region exon 4 sequences of six clinical cytomegalovirus strains were determined and compared with those of laboratory strains AD169 and Towne. Of 407 codons in exon 4, 33 (8.1%) showed interstrain variation at the peptide level and 74 (18%) showed interstrain variation at the nucleotide level. Variation occurred sporadically throughout the exon, and no grouping of strains was apparent. Published oligonucleotide primers proposed for diagnostic detection of cytomegalovirus by polymerase chain reaction have often been based on exon 4 sequences. Some of these primers show sequence mismatches with strains sequenced here. Amplification sensitivity for mismatched strains was reduced up to 100-fold. More-uniform detection sensitivity was achieved with primers of conserved sequence.


Detection of human cytomegalovirus in plasma of AIDS patients during acute visceral disease by DNA amplification. By using the polymerase chain reaction (PCR) amplification procedure, 19 (83%) of 23 plasma specimens obtained from individuals with AIDS and human cytomegalovirus (HCMV) visceral disease were found to be positive for plasma viremia as detected by PCR (PV-PCR), whereas 78% of cultures of peripheral blood leukocytes from the same samples were found to be positive. All 11 specimens prospectively obtained from individuals with acute HCMV disease were positive by PV-PCR. Plasma specimens from patients who received ganciclovir therapy rapidly became both culture and PV-PCR negative, and there was an excellent correlation between the two procedures. DNA detected by PV-PCR was unaffected by filtering plasma through a 0.2-microns-pore-size filter, although a conserved cellular gene, HLA-DQ alpha, was undetectable by PCR following filtration. HCMV DNA in plasma could be quantitated by PV-PCR by using endpoint serial dilutions, with detectable virus being present in 10(1) to 10(-2) microliters of plasma. A low titer of infectious virus could be detected in 2 of 11 plasma samples. The detection of HCMV DNA in plasma by PV-PCR promises to be a useful procedure for monitoring patients with AIDS suspected of having impending, acute, or recurrent HCMV visceral disease and suggests an additional route by which virus may disseminate in the immunocompromised host.


Biologically distinct subtypes of Mycobacterium avium differ in possession of insertion sequence IS901. Mycobacterium avium causes disease, principally tuberculosis in immunocompromised individuals. It is the most frequent cause of disseminated infections in AIDS patients in the West. The pathogen is also associated with disease in animals, chiefly birds and livestock, and may be isolated from environmental samples such as soil and water. Analysis of strains of M. avium isolated from clinical, veterinary, and environmental sources for the presence of the mycobacterial insertion sequences IS900 and IS901 demonstrates the specific association of IS901 to animal pathogenic M. avium strains. In contrast, most clinical M. avium strains and all AIDS-derived strains examined so far lacked IS901. Significant differences in the plasmid contents and serotypes of strains with and without IS901 were also found. We therefore suggest that the presence of IS901 divides M. avium into two clearly distinct subtypes with differing host range, virulence, plasmid possession, and serotyping antigens. By using DNA sequence data from IS901 and M. avium DNA, a set of polymerase chain reactions were developed for the specific detection and differentiation of these subtypes.


Immunoelectron microscopy for rapid diagnosis of varicella-zoster virus in a complicated case of human T-cell lymphotropic virus type 1 infection. Rapid techniques for the detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) are needed for optimal therapeutic management. VZV infection poses a serious threat, especially to seriously ill patients, for instance, immunocompromised patients. We report a case of human T-cell lymphotropic virus type 1-positive leukemia complicated by atypical multidermatomal herpes zoster. Viral culture and standard serological tests failed to prove VZV infection. Herpesvirus infection was confirmed by cytodiagnosis (Tzanck test). The final diagnosis of VZV was made by immunoelectron microscopy (IEM), which can differentiate between HSV and VZV. Immunoglobulin M antibodies in serum directed against VZV were detected by IEM but not by immunofluorescence. Because IEM was able to identify virus and analyze sera in only 2 h, it is considered a valuable additional tool for the rapid diagnosis of HSV and VZV infections.


Spectrum of monoclonal antibodies to coxsackievirus B-3 includes type- and group-specific antibodies. Fifteen monoclonal antibodies (MAbs) made to coxsackievirus B-3 were tested against a panel of enteroviruses by indirect immunofluorescence. The MAbs included seven which reacted with coxsackievirus B-3 only, five which reacted with more than one enterovirus included in the panel, one which reacted with broad enteroviral specificity and did not react with any heterologous virus (group specific), and two which reacted with all enteroviruses tested and at least one heterologous virus. The group-specific MAb identified 44 of 45 clinical isolates as enteroviruses, while the two broadly reactive MAbs reacted with all 45 of the clinical isolates. These MAbs are potentially important diagnostic reagents for grouping and typing enteroviruses by indirect immunofluorescence.


Mitochondrial oxidative stress after carbon monoxide hypoxia in the rat brain. To better understand the mechanisms of tissue injury during and after carbon monoxide (CO) hypoxia, we studied the generation of partially reduced oxygen species (PROS) in the brains of rats subjected to 1% CO for 30 min, and then reoxygenated on air for 0-180 min. By determining H2O2-dependent inactivation of catalase in the presence of 3-amino-1,2,4-triazole (ATZ), we found increased H2O2 production in the forebrain after reoxygenation. The localization of catalase to brain microperoxisomes indicated an intracellular site of H2O2 production; subsequent studies of forebrain mitochondria isolated during and after CO hypoxia implicated nearby mitochondria as the source of H2O2. In the mitochondria, two periods of PROS production were indicated by decreases in the ratio of reduced to oxidized glutathione (GSH/GSSG). These periods of oxidative stress occurred immediately after CO exposure and 120 min after reoxygenation, as indicated by 50 and 43% decreases in GSH/GSSG, respectively. The glutathione depletion data were supported by studies of hydroxyl radical generation using a salicylate probe. The salicylate hydroxylation products, 2,3 and 2,5-dihydroxybenzoic acid (DHBA), were detected in mitochondria from CO exposed rats in significantly increased amounts during the same time intervals as decreases in GSH/GSSG. The DHBA products were increased 3.4-fold immediately after CO exposure, and threefold after 120 min reoxygenation. Because these indications of oxidative stress were not prominent in the postmitochondrial fraction, we propose that PROS generated in the brain after CO hypoxia originate primarily from mitochondria. These PROS may contribute to CO-mediated neuronal damage during reoxygenation after severe CO intoxication.


Effect of streptozotocin-induced diabetes on GLUT-4 phosphorylation in rat adipocytes. We have examined the regulation of GLUT-4 phosphorylation in adipocytes isolated from diabetic rats. Despite progressive (40-70%) reductions in GLUT-4 protein contents on the 2nd, 7th, and 14th day of diabetes, the phosphorylation of GLUT-4 was increased two- to fourfold. These alterations were accompanied by concomitant reductions (40-66%) in the insulin-stimulated 2-deoxyglucose transport. Insulin treatment of diabetic animals for 5 d restored glucose transport activity, GLUT-4 protein, and GLUT-4 phosphorylation to control levels whereas vanadate and phlorizin were ineffective. In control adipocytes, insulin promoted GLUT-4 translocation from the low density microsomal (LDM) pool to the plasma membranes (PM) and decreased the state of GLUT-4 phosphorylation. In adipocytes isolated from the diabetic rats, insulin failed to stimulate GLUT-4 translocation and to decrease GLUT-4 phosphorylation. To explore the mechanism of the diabetes-induced increases in the GLUT-4 phosphorylation, we investigated phosphoserine phosphatase (PSPase) activities using 32P-labeled GLUT-4 and phosphorylase "a" as substrates. Diabetes resulted in 50-60% increase in the particulate PSPase activity and concomitant reductions in cytosolic PSPase activities. Although reduced cytosolic PSPase activity correlated with an inadequate dephosphorylation of LDM GLUT-4, the existence of highly phosphorylated PM GLUT-4 in the presence of increased particulate PSPase activity required additional explanation. To address this problem, we used PM GLUT-4 from diabetic rats as a substrate of particulate PSPase. Highly active diabetic particulate PSPase, which dephosphorylated control GLUT-4 and phosphorylase a, failed to dephosphorylate PM GLUT-4 from diabetic rats. These data suggest that PM GLUT-4 from diabetic rats is unable to interact with PSPase or that its phosphorylation sites are not accessible to PSPase action. In summary, an induction of diabetes with streptozotocin resulted in significant increases in GLUT-4 phosphorylation. In contrast to normal cells, insulin failed to promote GLUT-4 recruitment to the plasma membranes and its dephosphorylation in diabetic adipocytes. At the same time, diabetes appears to induce redistribution of PSPases, resulting in lower cytosolic activity and higher particulate activity. It also appears that the existence of highly phosphorylated GLUT-4 in the plasma membranes of diabetic adipocytes resulted from its inability to interact with particulate PSPases.


Human skin levels of retinoic acid and cytochrome P-450-derived 4-hydroxyretinoic acid after topical application of retinoic acid in vivo compared to concentrations required to stimulate retinoic acid receptor-mediated transcription in vitro. Metabolism of retinoic acid to a less active metabolite, 4-hydroxyretinoic acid, occurs via cytochrome P-450 isozyme(s). Effect of a pharmacological dose of retinoic acid on the level of retinoic acid in skin and on cytochrome P-450 activity was investigated. A cream containing 0.1% retinoic acid or cream alone was applied topically to adult human skin for four days under occlusion. Treated areas were removed by a keratome and a microsomal fraction was isolated from each biopsy. In vitro incubation of 3H-retinoic acid with microsomes from in vivo retinoic acid treated sites resulted in a 4.5-fold increase (P = 0.0001, n = 13) in its transformation to 4-hydroxyretinoic acid in comparison to in vitro incubations with microsomes from in vivo cream alone treated sites. This cytochrome P-450 mediated activity was oxygen- and NADPH-dependent and was inhibited 68% by 5 microM ketoconazole (P = 0.0035, n = 8) and 51% by carbon monoxide (P = 0.02, n = 6). Cotransfection of individual retinoic acid receptors (RARs) or retinoid X receptor-alpha (RXR-alpha) and a chloramphenicol acetyl transferase (CAT) reporter plasmid containing a retinoic acid responsive element into CV-1 cells was used to determine the ED50 values for stimulation of CAT activity by retinoic acid and its metabolites. Levels of all trans and 13-cis RA in RA-treated tissues were greater than the ED50 values determined for all three RARs with these compounds. Furthermore, the level of all trans RA was greater than the ED50 for RXR-alpha whereas the 4-OH RA level was greater than the ED50 for RAR-beta and RAR-gamma but less than for RAR-alpha and RXR-alpha. These data suggest that there are sufficient amounts of retinoic acid in treated skin to activate gene transcription over both RARs and RXR-alpha.


Cellular basis for blunted volume expansion natriuresis in experimental nephrotic syndrome. Experimental nephrotic syndrome results in sodium retention, reflecting, at least in part, an intrinsic defect in renal sodium handling in the distal nephron. We studied the relationships among plasma atrial natriuretic peptide (ANP) concentration, sodium excretion (UNaV), and urinary cyclic GMP excretion (UcGMPV) in vivo, and the responsiveness of isolated glomeruli and inner medullary collecting duct (IMCD) cells to ANP in vitro, in rats with adriamycin nephrosis (6-7 mg/kg body weight, intravenously). 3-5 wk after injection, rats were proteinuric and had a blunted natriuretic response to intravenous infusion of isotonic saline, 2% body weight given over 5 min. 30 min after onset of the infusion, plasma ANP concentrations were elevated in normals and were even higher in nephrotics. Despite this, nephrotic animals had a reduced rate of UcGMPV after the saline infusion, and accumulation of cGMP by isolated glomeruli and IMCD cells from nephrotic rats after incubation with ANP was significantly reduced compared to normals. This difference was not related to differences in binding of 125I-ANP to IMCD cells, but was abolished when cGMP accumulation was measured in the presence of 10(-3) M isobutylmethylxanthine or zaprinast (M&B 22,948), two different inhibitors of cyclic nucleotide phosphodiesterases (PDEs). Infusion of zaprinast (10 micrograms/min) into one renal artery of nephrotic rats normalized both the natriuretic response to volume expansion and the increase in UcGMPV from the infused, but not the contralateral, kidney. These results show that, in adriamycin nephrosis, blunted volume expansion natriuresis is associated with renal resistance to ANP, demonstrated both in vivo and in target tissues in vitro. The resistance does not appear related to a defect in binding of the peptide, but is blocked by PDE inhibitors, suggesting that enhanced cGMP-PDE activity may account for resistance to the natriuretic actions of ANP observed in vivo. This defect may represent the intrinsic sodium transport abnormality linked to sodium retention in nephrotic syndrome.


Inhibition of apical Na+ channels in rabbit cortical collecting tubules by basolateral prostaglandin E2 is modulated by protein kinase C. We used the cell-attached patch clamp technique to investigate the interaction of exogenous prostaglandins (PG), intracellular [Ca2+]i, and protein kinase C (PKC) on the high selectivity, 4 pS Na+ channel found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) cultures grown on collagen supports with 1.5 microM aldosterone. Application of 0.5 microM PGE2 to the basolateral membrane decreased mean NP0 (number of channels times the open probability) for apical Na+ channels by 46.5% (n = 9). There was no consistent change in NP0 after apical 0.5 microM PGE2 (n = 12) or after apical or basolateral 0.5 microM PGF2 alpha (n = 8). Release of [Ca2+]i stores with 0.25 microM thapsigargin (n = 7), or activation of apical membrane PKC with apical 0.1 microM 4 beta-phorbol-12-myristate-13-acetate (n = 5) or 10 microM 1-oleyl-2-acetylglycerol (n = 4) also decreased NP0. Depletion of [Ca2+]i stores (0.25 microM thapsigargin pretreatment) (n = 7) or inhibition of apical PKC (100 microM D-sphingosine pretreatment) (n = 8) abolished the inhibitory effects of basolateral PGE2. (a) apical Na+ transport in rabbit CCT principal cells is modulated by basolateral PGE2; (b) the mechanism involves release of IP3-sensitive, [Ca2+]i stores; and (c) Ca(2+)-dependent activation of apical membrane PKC, which then inhibits apical Na+ channels.


Cellular responses to steroids in the enhancement of Na+ transport by rat collecting duct cells in culture. Differences between glucocorticoid and mineralocorticoid hormones. It has recently been discovered that both mineralocorticoid (MC) and glucocorticoid (GC) hormones can stimulate electrogenic Na+ absorption by mammalian collecting duct cells in culture. In primary cultures of rat inner medullary collecting duct (IMCD) cells, 24-h incubation with either MC or GC agonist stimulates Na+ transport approximately threefold. We have now determined that the effects were not additive, but the time courses were different. As aldosterone is known to stimulate citrate synthase, Na+/K+ ATPase activity, and ouabain binding in cortical collecting duct principal cells, we determined the effects of steroids on these parameters in IMCD cells. MC and GC agonists both produced a small increase in citrate synthase activity. There was no increase in Na+/K+ ATPase activity but specific ouabain binding was increased more than two-fold by either agonist. To determine the role of apical Na+ entry in the steroid-induced effects, the Na+ channel inhibitor, benzamil, was used. Benzamil did not alter the stimulation of citrate synthase activity by either steroid. In contrast, GC stimulation of ouabain binding was prevented by benzamil, whereas MC stimulation was not. We conclude that there are differences in the way that MC and GC hormones produce an increased Na+ transport. Both appear to produce translocation (or activation) of pumps into the basolateral membrane. GC stimulation of pump translocation requires increased Na+ entry whereas MC stimulation does not.


Renal tubular responsiveness to atrial natriuretic peptide in sodium-retaining chronic caval dogs. A possible role for kinins and luminal actions of the peptide. 60% of chronic caval dogs with ascites did not respond to atrial natriuretic peptide (ANP) (75 ng.kg-1.min-1) with a natriuresis (TIVC-NR; delta UNaV = 2 +/- 0.8 mu eq/min) whereas the remaining 40% responded normally (TIVC-R; delta UNaV = 216 +/- 50 mu eq/min). Since proximal tubule neutral endopeptidase 24:11 (NEP) destroys most of intrarenal luminal ANP and kinins, we attempted to convert TIVC-NR into TIVC-R by providing NEP inhibition with SQ 28603 at 30 mg/kg. This potent and specific NEP inhibitor produced a natriuresis when administered alone to nine TIVC-NR dogs (delta UNaV = 67 +/- 2 mu eq/min) and permitted a natriuresis in the presence of ANP (delta UNaV = 97 +/- 18 mu eq/min). A natriuretic response to ANP could also be induced in TIVC-NR dogs by providing renal arterial bradykinin or intravenous captopril, a kininase inhibitor. Urodilatin, a natriuretic peptide not destroyed by intrarenal NEP was without effect in TIVC-NR dogs but increased UNaV when given to TIVC-R and normal dogs. Providing bradykinin to TIVC-NR now permitted an increment in delta UNaV (62 mu eq/min) when urodilatin was reinfused. TIVC-R dogs could be converted into TIVC-NR by pretreating with a specific bradykinin antagonist before infusing ANP. We conclude that TIVC-NR dogs are deficient in intrarenal kinins but are converted to responding dogs after NEP inhibition because of increased kinin delivery to the inner medullary collecting duct.


Endothelin inhibits vasopressin-stimulated water permeability in rat terminal inner medullary collecting duct. Renal tubule solute and water transport is subject to regulation by numerous factors. To characterize direct effects of the recently discovered peptide endothelin (ET) on renal tubule transport, we determined signaling mechanisms for ET effects on vasopressin (AVP)-stimulated water permeability (PF) in rat terminal inner medullary collecting duct (IMCD) perfused in vitro. ET caused a rapid, dose-dependent, and reversible fall in AVP- but not cyclic AMP-stimulated PF, suggesting that its effect on PF is by inhibition of cyclic AMP accumulation. Indomethacin did not block ET actions, ruling out a role for prostaglandins in its effect. The protein kinase C (PKC) inhibitor calphostin, or pretreatment of perfused tubules with pertussis toxin, blocked ET-mediated inhibition of AVP-stimulated PF. ET caused a transient increase in intracellular calcium ([Ca2+]i) in perfused tubules, an effect unchanged in zero calcium bath or by PT pretreatment. ET effects on PF and [Ca2+]i desensitized rapidly. Inhibition of PF was transient and largely abolished by 20 min ET preexposure, and repeat exposure to ET did not alter [Ca2+]i. In contrast, PGE2-mediated inhibition of AVP-stimulated PF and increase of [Ca2+]i were sustained and unaltered by prior exposure of IMCD to ET. Thus desensitization to ET is homologous. We conclude that ET is a potent inhibitor of AVP-stimulated water permeability in rat terminal IMCD. Signaling pathways for its effects involve both an inhibitory guanine nucleotide-binding protein and phospholipase-mediated activation of PKC. Since ET is synthesized by IMCD cells, this peptide may be an important autocrine modulator of renal epithelial transport.


Effects of uninephrectomy on electrical properties of the cortical collecting duct from rabbit remnant kidneys. Microelectrode techniques were used to determine the Na+ and K+ transport properties of the collecting duct cell in the isolated cortical collecting duct (CCD) from rabbits 14 d after uninephrectomy (UNX); results were compared with those from sham-operated rabbits (control). UNX had no effects on plasma aldosterone levels. The CCDs from UNX rabbits exhibited structural hypertrophy. The lumen negative transepithelial voltage and the basolateral membrane voltage (VB) were elevated in the UNX group. Although the transepithelial conductance (GT) and the fractional apical membrane resistance (fRA) were not different between the two groups, the conductances of the apical and the basolateral membranes were increased, and the tight junction conductance was decreased in the UNX group. The amiloride-sensitive changes in apical membrane voltage (VA), fRA, and GT were greater in the UNX group. The changes in VA upon raising the perfusate K+ concentration and the changes in VA and GT upon addition of Ba2+ to the perfusate were elevated in the UNX group. Upon raising K+ in the bath, a large depolarization of VB was observed in the UNX group. Lowering the bath Cl- resulted in a small depolarization of VB in the UNX group. Addition of Ba2+ to the bath in the UNX group caused the VB to hyperpolarize in parallel with decreases in GT and fRA whereas in the control group it had no effect on VB. Addition of ouabain to the bath resulted in a large depolarization of VB in the UNX group. We conclude that (a) UNX stimulates conductances of Na+ and K+ in the apical membrane, active Na(+)-K+ pump activity, and K+ conductance in the basolateral membrane, independently of plasma aldosterone; (b) The basolateral membrane in the tubules of UNX rabbits is more selective to K+; and (c) the hyperpolarization of VB upon UNX may increase passive K+ entry into the cell across the basolateral membrane.


Expression of transforming growth factor-beta 1 is increased in human vascular restenosis lesions. Human atheromata obtained in vivo were used to test the hypothesis that transforming growth factor-beta 1 plays a role in the development of vascular restenosis. We analyzed 28 specimens from patients with primary atherosclerotic or restenotic lesions; 26 of these were obtained by directional atherectomy and 2 at the time of coronary bypass surgery. Seven control tissues included operatively excised segments of human internal mammary artery, myocardium, and unused portions of vein graft obtained intraoperatively. From these 35 specimens, 210 sections were examined using in situ hybridization. Measurement of silver grains/nucleus disclosed that expression of transforming growth factor-beta 1 mRNA was highest in restenotic tissues (P < 0.001 vs. primary atherosclerotic tissues) and lowest in nonatherosclerotic (control) tissues. In cultures of human vascular smooth muscle cells grown from explants of internal mammary artery, expression of mRNA for transforming growth factor-beta 1 was significantly greater in subconfluent than in confluent smooth muscle cells (P = 0.05). Transforming growth factor type-beta III receptor was expressed in cell cultures and undetectable in the tissue specimens. Sections taken adjacent to those studied by in situ hybridization were examined by immunohistochemistry using antibodies against transforming growth factor-beta 1 and alpha-actin (as a marker for smooth muscle cells) and disclosed transforming growth factor-beta 1 in smooth muscle cells present in these sections. These findings are consistent with the concept that transforming growth factor-beta 1 plays an important role in modulating repair of vascular injury, including restenosis, after balloon angioplasty.


Microfilament-dependent activation of Na+/K+/2Cl- cotransport by cAMP in intestinal epithelial monolayers. cAMP-mediated stimulation of Cl- secretion in the human intestinal cell line T84 is accompanied by significant remodeling of F-actin, and both the secretory and cytoskeletal responses may be largely ablated by previous cell loading with phalloidin derivatives, reagents that prevent dynamic reordering of microfilaments (1991. J. Clin. Invest. 87:1903-1909). In this study, we examined the effect of phalloidin loading on the cAMP-elicited activity of the individual membrane-associated transport proteins involved in electrogenic Cl- secretion. Efflux of 125I and 86Rb was used to assay forskolin-stimulated Cl- and K+ conductances, respectively, and no inhibitory effect of phalloidin could be detected. Na+/K(+)-ATPase pump activity, assessed as bumetanide-insensitive 86Rb uptake and the ability of monolayers to generate a Na+ absorptive current in response to apical addition of a Na+ ionophore, was not different between control and phalloidin-loaded monolayers. Forskolin was found to stimulate Na+/K+/2Cl- cotransport (bumetanide-sensitive 86Rb uptake) in time-dependent fashion. In the absence of any agonist, cotransporter activity was markedly decreased in phalloidin-loaded monolayers. Furthermore, under phalloidin-loaded conditions, the forskolin-elicited increase in bumetanide-sensitive 86Rb uptake was markedly attenuated. These findings suggest that cAMP-induced activity of Cl- channels, K+ channels, and the Na+/K(+)-ATPase are not influenced by F-actin stabilization. However, cAMP-induced activation of the Na+/K+/2Cl- cotransporter appears to be microfilament-dependent, and ablation of this event is likely to account for the inhibition of cAMP-elicited Cl- secretion seen in the phalloidin-loaded state. Such findings suggest that Na+/K+/2Cl- cotransporter is functionally linked to the cytoskeleton and is a regulated site of cAMP-elicited electrogenic Cl- secretion.


Thrombin receptor expression in normal and atherosclerotic human arteries. Thrombin is a multifunctional serine protease generated at sites of vascular injury. A host of thrombin actions on vascular endothelial cells, smooth muscle cells, and macrophages has been defined in cell culture systems, but the in vivo significance of these activities is unknown. We have defined the expression of the recently identified receptor for thrombin in human arteries by both in situ hybridization and immunohistochemistry. In normal-appearing arteries, thrombin receptor was expressed almost exclusively in the endothelial layer. By contrast, in human atheroma, the receptor was widely expressed, both in regions rich in macrophages and in regions rich in vascular smooth muscle cells and mesenchymal-appearing intimal cells of unknown origin. Thrombin receptor was expressed by human vascular endothelial cells and smooth muscle cells in culture and by macrophages obtained by bronchioalveolar lavage, thus demonstrating that all three cell types are indeed capable of expressing the thrombin receptor. These results establish thrombin receptor activation as a candidate for contributing to sclerotic and inflammatory processes in the human vasculature, such as those that occur in atherosclerosis and restenosis.


Morbidity associated with removal of the submandibular gland. Postoperative complications in 206 submandibular gland excisions, excluding those resulting from benign or malignant tumours, carried out during a 15-year period were reviewed. Most patients (62%) had sialolithiasis. Coexistence of sialolithiasis and nephrolithiasis was documented in 5.5% of cases. Early postoperative complications (particularly infection) developed in 14.6% of the cases, whereas late complications appeared in 25.3% of the cases (residual inflammation in Wharton's duct 7.3%). Neurological complications were observed in 16% of the cases. In 7 cases (3.4%) several nerves were involved and almost always the hypoglossal nerve. In 37.4% of the cases, these lesions resolved spontaneously in a mean period of 4 months. In those cases with a permanent neurological deficit, the facial nerve was the most often affected (7.7%) followed by the hypoglossal (2.9%) and the lingual nerve (1.4%). A single case of gustatory sweating (Frey's) syndrome was observed.


Optic neuropathy: a rare paraneoplastic syndrome. A 63-year-old man developed gradually progressive bilateral loss of vision, cerebellar ataxia, and downbeat nystagmus. Visual acuity was 20/400 OD and 20/200 OS, with cecocentral scotomas OU. Fundus examination showed bilateral optic atrophy and a vitreous cellular reaction. MRI of the brain was normal. CSF protein was elevated, with increased IgG levels but no malignant cells. Biopsy of a pulmonary lymph node showed undifferentiated small cell carcinoma. Neoplastic cells were positive for neuron-specific enolase. Serum contained IgG, which reacted with neuronal and glial cytoplasm and processes. IgG reactivity with systemic tissues and the patient's tumor was not different from that observed with control sera. Paraneoplastic optic neuropathy should be considered in patients with unexplained visual loss and malignancy, and our observations suggest a possible immunologic basis for this condition.


Color flow Doppler ultrasonography: comparison with peripheral arteriography for the investigation of peripheral vascular disease. Using arteriography as the gold standard, Color flow Doppler ultrasonography was evaluated with regard to its ability to detect peripheral vascular occlusive disease and hemodynamically significant stenosis in patients having peripheral arteriography. One hundred legs in 51 patients were compared at seven arterial segments for disease. Color flow Doppler ultrasonography correctly detected 84 occluded segments, and demonstrated a sensitivity and specificity for patency vs occlusive disease of 95% and 99%, respectively. One hundred and thirty hemodynamically significant lesions (occlusions plus significant stenosis) were correctly identified with color flow Doppler ultrasonography, with a sensitivity and specificity of 92% and 97%, respectively. Color flow Doppler ultrasonography is a safe, inexpensive, and noninvasive method of accurately documenting significant peripheral arterial disease and offers a new first-line investigation for patients presenting with symptoms of peripheral arterial insufficiency.


Ultrasonic detection of viscera slide as an indicator of abdominal wall adhesions. Real-time ultrasonography can detect the movement of viscera immediately deep to the abdominal wall. This motion of abdominal contents is called viscera slide, and is produced by the force of respiratory motion (spontaneous viscera slide) or by manual ballottement of the abdomen (induced viscera slide). Viscera slide was observed in 18 "normal" subjects (no history of previous abdominal surgery or peritonitis) and in 24 subjects at "risk" for abdominal wall adhesions because of previous abdominal operations or past history of peritonitis. In 14 of the 24 "risk" group subjects, spontaneous and induced viscera slide was restricted to excursions of less than 1 cm (58.3%). Operations were performed on 18 patients, which confirmed the fact that restriction of ultrasonically detected viscera slide identified abdominal wall adhesions in all cases, but no adhesions were found in patients with normal viscera slide. This ultrasonic finding of restricted viscera slide may be useful in the preoperative discovery and localization of abdominal wall adhesions prior to laparoscopy or laparotomy.


Growth standards for anatomic measurements and growth rates derived from longitudinal studies of normal fetal growth. A statistical procedure for deriving growth standards for anatomic measurements and their growth rates from longitudinal studies of fetal growth was evaluated using Rossavik growth models for the biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur diaphysis length (FDL) determined in a previous study of normal fetal growth. For each anatomic parameter, the coefficients c and s of the model was used to define a set of growth curves that constituted the boundary growth curves of a region containing 95% of the growth curves of this data set. The set of boundary growth curves was used to specify the mean, lower limit, and upper limit values for the anatomic parameter and its growth rate at weekly intervals between 14 and 38 weeks, menstrual age. Comparison of these values to those determined from cross-sectional studies of fetal growth gave differences of -1.9% to 4.8% (SD: +/- 0.9 to +/- 2.6) for mean vs. predicted value of the anatomic measurements. For the lower limit, similar values were 0.4% to 13.8% (SD: +/- 1.7 to +/- 8.8); for the upper limit the values were 8.3% to 18.0% (SD: +/- 1.5 to +/- 7.0). Comparisons of HC growth rates determined using polynomial and Rossavik growth models gave values of -3.4% (SD: +/- 4.4) for mean vs. predicted value, -12.6% (SD: +/- 10.6) for the lower limit and 5.2% (SD: +/- 9.3) for the upper limit. The degree of agreement was similar for AC growth rates.(ABSTRACT TRUNCATED AT 250 WORDS)


Ultrasound imaging in tropical pancreatitis. Tropical pancreatitis differs in many respects from the chronic pancreatitis seen in Western countries. The present study was carried out to evaluate the role of ultrasonography in the diagnosis of tropical pancreatitis (TP) and to characterize the ultrasound findings in tropical pancreatitis. Patients referred with a suspected diagnosis of tropical pancreatitis formed the subjects for the study. Plain x-rays of the abdomen, ultrasonography, and endoscopic retrograde cholangio-pancreatography (ERCP) were carried out in all cases. Of the 25 cases, 17 patients had ERCP evidence of pancreatitis. Duct dilatation (82%) and demonstration of calculi were the most common ultrasound findings. Pancreatic atrophy (53%) was also a major feature of TP. Compared with ERCP, ultrasonography had a sensitivity of 94% and a specificity of 100%. Only one case with mild changes in ERCP was missed by ultrasonography. For the diagnosis and planning of surgery in TP, ultrasonography can replace ERCP. Even complications like cysts and malignancies are detected by ultrasonography.


Incidentally detected renal cell carcinoma: role of ultrasonography. The widespread use of modern diagnostic imaging techniques, especially computed tomography and sonography, has led to the detection of an increasing number of serendipitous renal neoplasms, with no signs nor symptoms related to the neoplastic renal disease. In the last 4 years, 54 consecutive patients (20 females and 34 males) with renal cell carcinoma were reviewed. In 26 patients (48.15 per cent) the diagnosis was made as an incidental finding by an abdominal ultrasound examination. Clear signs and symptoms related to the neoplastic disease were present in 28 cases (51.85 per cent). The pathologic type of each neoplasm was categorized, and each carcinoma was staged. In the patients not surgically treated, the stage was determined on the basis of the diagnostic imaging reports. The results of this study suggest that the incidentally detected tumors are of a significantly lower stage than the symptomatic tumors (Wilcoxon rank sum test: P less than 0.0073 and X2 test: P less than 0.013). Early detection of renal tumors may improve the prognosis and the overall survival of patients with renal cell carcinoma and allow one to plan radical or partial nephrectomy, since local extension has a considerable impact on the operative strategy. Our experience emphasizes the role of ultrasound in the increased early detection of renal cell carcinoma.


Renal transplantation: the normal morphological and Doppler ultrasound examination. If ultrasound imaging and image-directed Doppler examination of the renal allograft is to provide information with respect to graft complications and graft failure, then a clear understanding of normal characteristics is required. Criteria for abnormality of a graft will include change in size, in parenchymal pattern, and in blood flow characteristics in the main and branch renal arteries and within the renal vein. Normal measurements for the renal allograft are presented as well as morphological and numerical data relating to cortico medullary differentiation. A wide range of values for indices quantifying Doppler data is recorded in the literature. Mean values and standard deviations from the mean are presented. The importance of different parameters as potential indicators of disease are stressed, but the limitations, usually as a result of normal variation and lack of specificity, are presented. The normal features of color-coded Doppler vascular supply, perfusion, and drainage are described.


Doppler and color Doppler imaging in acute transplant failure. Most renal transplants are lost by rejection. A method is required to identify and discriminate between this and acute tubular necrosis and cyclosporin toxicity. The sonogram of the normal renal transplant is characteristic. Early Doppler studies measuring a rise in vascular impedance in acute rejection showed a high sensitivity and specificity. This appears, at least in part, to have been due to patient selection. We conclude that Doppler studies cannot be used to differentiate between the main parenchymal causes of renal transplant failure, although it can be helpful, with other clinical information, in supporting a diagnosis and in monitoring the effects of treatment.


Vascular complications in the adult kidney transplant recipient. Vascular complications of renal transplantation occurred in 15% of the cases. They are thrombotic infarct, arterial stenosis, arterio-venous fistula, and chronic arterial diseases. From 900 renal transplantations performed, only 120 (made since 1989) were studied with color flow Doppler (CFD). Lack of arterial signal is indicative of main arterial thrombosis (or of renal infarct if thrombosis is limited). At the site of arterial stenosis, high velocity and turbulence are found. If the stenosis is more than 70%, the rising systolic time is longer than 0.07 sec in the post-stenotic artery. Arterio-venous fistulas are frequent after renal biopsy. They provoke vibrations transmitted to peri-vascular tissues and seen with CFD as a large area of turbulence. In the feeding artery, Fast Fourier Transformation (FFT) showed a high velocity with a low resistive index and pulsed flow in the outgoing vein. Chronic arterial diseases include cyclosporine A intoxication and chronic rejection. These two diseases cannot be diagnosed by CFD alone.


Morphological appearance of renal allografts in transplant failure. Renal allograft dysfunction may be due to a variety of causes including acute and chronic rejection, acute tubular necrosis, cyclosporine A toxicity, glomerulonephritis, and surgical causes. It can be difficult to distinguish between these etiologies. Ultrasound is a relatively simple, noninvasive method for imaging the transplant kidney, and is excellent for the diagnosis of surgical complications. The features of rejection include an increase in renal volume, reduced echogenicity, indistinctness of the corticomedullary junction, and splaying of the medullary pyramids. These findings, although suggestive of rejection, cannot differentiate reliably between rejection and other causes of allograft hypofunction. Doppler studies may show an increased resistive index in a kidney undergoing rejection, but this finding may also be seen in other pathological processes and is not specific. A normal ultrasound examination does not exclude rejection. Ultrasound is useful as a baseline examination. Abnormal findings should be interpreted in the light of the clinical situation in each case. Where there is any doubt about the diagnosis, ultrasound-guided biopsy should be performed.


Doppler and color Doppler ultrasonography in renal transplants: chronic rejection. Although Doppler and color Doppler ultrasonography has been used extensively to examine acute changes in renal allograft function, there have been few reports on the role of Doppler ultrasonography in the assessment of chronic rejection. In those studies that have addressed chronic rejection, there is agreement that changes in renal function are not accompanied by consistent changes in flow wave-form shape. The pathology of chronic rejection may be very different from the changes seen in acute rejection and its onset is usually gradual. Some authors have suggested that the decline in function is accompanied by lower Doppler frequency shifts (corresponding to lower velocities) in intra-renal arteries. In a preliminary study comparing grafts with normal renal function and with biopsy-proven chronic rejection, it has been shown that for grafts with chronic rejection, observed intra-renal velocities are lower, especially at segmental and interlobar levels. This may be a more reliable reflection of reduced renal blood flow in chronic rejection than conventional Doppler ultrasound analysis of flow waveform shape. However, the diagnostic use of changes to intra-renal flow velocities in chronic rejection is currently very limited.


Complications of renal transplantation and the role of interventional radiology. As rejection therapy has improved, the interventional complications of renal transplantation have increased in relative importance as potentially reversible causes of graft malfunction and failure. Ultrasonically guided aspiration and drainage techniques are essential for the characterization and subsequent management of perinephric fluid collections that occur in as many as 51% of cases, of which up to 18% may be clinically significant. In a series of 507 consecutive renal transplants, urological complications were seen in 9% of patients, consisting of ureteric obstruction in 6% and urinary leaks in 3%. Detection via ultrasound scanning, evaluation via antegrade pyelography and pressure-flow tests, and management via percutaneous nephrostomy and ureteric stenting all contribute to rapid diagnosis and subsequent treatment. Serial ultrasound scanning of renal transplants and the aggressive use of interventional radiological techniques are vital to the early detection and management of complications, and can lead to preservation of graft function and continued improvement in graft survival figures.


Ultrasound assessment of the problem kidney transplant: a surgical prospective. Ultrasound investigation contributes to the assessment of transplanted kidneys. Ureteric obstruction and peri-transplant fluid collections are shown satisfactorily, but the differentiation of graft rejection from cyclosporin A nephrotoxicity and continuing acute tubular necrosis is less straightforward. Doppler ultrasound is used to detect changes in graft blood circulation, but the results of such assessment vary between centers, and further improvements are required to improve the accuracy of diagnosis of causes of graft impairment by noninvasive methods.


Ultrasonography of the native kidney in dialysis and transplant patients. The native kidneys in patients on dialysis or after transplantation tend to be overlooked until problems occur in relation to them. Their appearance can be variable and does not bear any consistent relationship to the cause of the renal failure; although in some cases, such as polycystic kidney disease, there are specific changes to be seen. Size can also be variable with little correlation to pathology in most cases. Acquired cystic disease of the kidneys is seen in up to 92% of long-term dialysis patients and also can be seen in patients with chronic renal failure. Proliferative changes occur in the kidneys which result in the development of cysts, adenomas and, in approximately 1% to 2% of dialysis patients, malignant lesions. It is probable that these changes are caused by a combination of factors, including circulating agents which are not cleared adequately by dialysis. There is some evidence that these changes are halted, or even reversed following a successful transplant. Problems with infection and hemorrhage may occur in patients with polycystic kidney disease. Problems with infection may also occur in patients with calculi, with longstanding pyelonephritis, or reflux.


Cytogenetic characterisation of small round cell tumours using fine needle aspiration. Cytogenetic analysis of short term cultures of fine needle aspirates from two tumours, characterised cytologically as small round cell neoplasms, showed specific clonal chromosomal abnormalities. In both cases the cytogenetic finding of a t(11; 22) (q24; q12) helped determine their diagnosis as peripheral neuroectodermal tumours of the thoraco-pulmonary region (Askin's tumour). These findings suggest that cytogenetics can reliably distinguish neoplasms which present as undifferentiated small round cell tumours.


Human herpes virus 6 and endogenous biotin in salivary glands. To detect the presence of human herpes virus 6 (HHV6) and endogenous biotin in paraffin wax embedded and frozen salivary glands. Two stage indirect and streptavidin-biotin immunoperoxidase techniques were used to visualise the antigens. HHV6 could not be shown in any of the tissues. However, considerable endogenous biotin antigenicity was detected in the glandular elements of the paraffin wax embedded material. Results obtained with avidin-biotin detection systems should be interpreted with caution, especially when glandular epithelium is being stained. This may apply to both immunoperoxidase and in situ hybridisation techniques. The use of an anti-biotin antibody as a standard control should be considered.


Immunocytochemical localization of DARPP-32, a dopamine and cyclic-AMP-regulated phosphoprotein, in the primate brain. The localization of DARPP-32, a dopamine and cAMP-regulated phosphoprotein, has been studied in monkey brain by immunocytochemistry. This study indicates that DARPP-32 is enriched in neurons in regions receiving a dense dopamine input from the substantia nigra and ventral tegmental area. Thus, the majority of somata in the anterior olfactory area, nucleus accumbens, caudate nucleus, and putamen are immunoreactive for DARPP-32. In the caudate nucleus, immunoreactive spines receive asymmetric contacts from unlabeled axon terminals. Immunoreactive somata have diameters of 10-15 microns. In regions known to receive projections from these nuclei, immunoreactivity is confined to small puncta that represent axons and axon terminals. Regions in which immunoreactivity is present in puncta include the ventral pallidum, globus pallidus, and substantia nigra pars reticulata. Dopaminergic neurons themselves are not immunoreactive. Neurons containing moderate to weak immunoreactivity for DARPP-32 are observed in portions of the cerebral cortex, particularly in the temporal cortex (layer VI). DARPP-32-positive neurons are also present in the cerebellum, in the medial habenula, and in portions of the bed nucleus of the stria terminalis and amygdaloid complex. DARPP-32 immunoreactivity is also present in astrocytes in the subcortical white matter and in tanycytes in the arcuate nucleus and median eminence. DARPP-32 may be an effective marker for dopaminoceptive neurons in which the actions of dopamine on the D-1 dopamine receptor are mediated through cAMP and its associated protein kinase.


Relationships between dendritic morphology and cytochrome oxidase compartments in monkey striate cortex. In primate striate cortex, staining for the mitochondrial enzyme cytochrome oxidase reveals a regular pattern of intense staining, the blobs, which are surrounded by the lighter stained interblob regions. Neurons in both compartments exhibit profound functional differences: blob cells have color selective, unoriented receptive fields, whereas interblob cells are usually not color selective and have oriented receptive fields. Neuroanatomical tracing studies have shown that blob and interblob cells receive different inputs and participate in different projections. It is not known, however, whether this compartmental organization is also reflected in the dendritic morphology of individual cells. We therefore combined intracellular staining with cytochrome oxidase histochemistry to study the relationship between cell morphology and blob pattern in layers 2 and 3 of macaque striate cortex. Single cells were injected with the fluorescent dye lucifer yellow in lightly fixed tangential sections. Adjacent sections were reacted for cytochrome oxidase to reveal the blobs. The spatial relationship between stained cells and the pattern of the blobs were subsequently determined by aligning the sections by using radially running blood vessels as landmarks. Our results show that pyramidal cells located in blob and interblob regions do not differ in their soma size, spine density, and basal dendritic field structure. This indicates that the characteristic functional properties of the neurons in both compartments do not depend on the morphology of their dendritic trees. Since the elongation of the dendritic fields of blob and interblob cells was also found to be similar, we conclude that cortical orientation selectivity is not generated through elongated dendritic fields. We found several cells with dendrites freely crossing the borders between blob and interblob regions. These cells might correspond to cells with "mixed" receptive field properties, e.g., color selective oriented cells, which in physiological studies were found at the transition from blob to interblob regions. However, there were also a number of cells that respected the borders. A quantitative analysis of the dendritic fields revealed that 67% of the cells located close to the borders have a tendency to confine their dendrites to only one compartment. Thus the pattern of basal dendrites of these cells might be shaped by the parcellation of the striate cortex in blob and interblob regions. These dendritic field asymmetries may help to maintain the segregation at the single cell level into different processing channels in monkey striate cortex.


Inhibition of adrenergic proton extrusion in rainbow trout red cells by nitrite-induced methaemoglobinaemia. The effects of nitrite-induced methaemoglobinaemia on adrenergic proton extrusion from rainbow trout red blood cells were studied using the pH-stat method. In control conditions adrenergic proton extrusion was completely inhibited by amiloride and was greater in deoxygenated than in oxygenated erythrocytes. Nitrite-induced methaemoglobinaemia was associated with a pronounced reduction in the catecholamine-stimulated proton efflux from both deoxygenated and oxygenated erythrocytes. In deoxygenated erythrocytes the initial proton efflux upon catecholamine stimulation decreased by 60-70%, while the percentage of methaemoglobin in the red cells increased from the control level of 1-3% to 20%. In oxygenated erythrocytes the decrease was 30% at the same methaemoglobin percentage range. It is suggested that the pronounced influence of nitrite-induced methaemoglobinaemia on adrenergic proton efflux results from an inhibition of the red cell sodium/proton exchanger by the R-like haemoglobin conformations.


Swollen keratinocytes: a histologic marker of unusual human papillomavirus-type infection and immunosuppression. Cutaneous papillomavirus infection is common in patients who are immunosuppressed. We describe swollen keratinocytes in the granular layer in lesions from four patients who had human immunodeficiency virus infection. These cells were similar to those described in skin lesions of epidermodysplasia verruciformis. Amplification of DNA from the lesions revealed an amplimer for human papillomavirus using a consensus primer for a highly conserved region of the L1 open reading frame; however, specific binding was not noted when radiolabelled probes for human papillomavirus types 6, 11, 16, 18, and 33 were used. We conclude that the presence of these distinctive swollen cells strongly suggests immunosuppression and quite possibly infection by a less common papillomavirus type.


Eccrine syringofibroadenoma (Mascaro): an ultrastructural study. To confirm the eccrine acrosyringeal differentiation of eccrine syringofibroadenoma (ESFA) and to elucidate the histogenesis of its angiofibrotic stroma, a case of ESFA from a 45-year-old man was examined by light and electron microscopy. Histologically, the parenchyma featured anastomosing, slender epithelial cords containing small cuboidal cells and occasional duct-like structures. The stroma had increased numbers of mast cells, increased capillaries with swollen endothelial cells, and prominent fibrosis. Ultrastructurally, the following findings were characteristic of ESFA: a) abundant glycogen particles in epithelial cells, b) numerous intracytoplasmic and extracellular spaces lined with microvilli, c) intraepithelial duct formation, consisting of microvilli, vesicles, rod-shaped dense bodies, multivesicular dense bodies, and peripheral network of tonofilaments, and d) large numbers of mast cells, closely associated with fibroblasts, surrounding increased numbers of capillaries containing swollen endothelial cells. These ultrastructural features support the acrosyringeal differentiation of ESFA. We hypothesize that mast cell hyperplasia and degranulation may play an important role in the formation of the angiofibrotic stroma.


Different bacteriophage resistance mechanisms in Streptococcus salivarius subsp. thermophilus. Streptococcus salivarius subsp. thermophilus strain NST5 exhibited a temperature-dependent defence mechanism against the virulent bacteriophages phi B1.2 and phi A1.1. It was active at 42 degrees C but not at 30 degrees C as demonstrated by a significant increase of both plaque size and efficiency of plaquing. This defence mechanism did not affect host-dependent phage replication and did not interfere with phage adsorption to NST5. These results suggest that it interfered with phage development. The phages phi T33, phi T58, phi D1, phi T21 and phi T9, belonging to the same phage type as phi B1.2, were examined for their ability to infect NST3 and NST5. Restriction modification systems of different specificity were detected in NST3 and NST5; host-dependent phage replication was detected at 30 and 42 degrees C; an abortive defence mechanism was detected in NST5 which was active at 42 degrees C, but not 30 degrees C, and was independent of restriction modification action or interference with phage adsorption. Our investigations of phage-host interactions showed that the two Str. salivarius subsp. thermophilus strains studied avoided attack by related bacteriophages by evolving at least three different resistance systems.


Application of theoretically optimal sampling schedule designs for fiber digestion estimation in sacco. Three different geometrically spaced sampling schedule designs, a theoretically optimal design, and a design that included all sampling times were evaluated by comparing parameter estimates, half-life, R2, and an indicator of variance-covariance space. Alfalfa and oat hays were tested using nylon bags placed in the rumen of a fistulated, non-lactating cow, and the amount of NDF remaining was measured at specified times. Parameters were estimated from f(t, phi) = Ae-K(t-lag) + U, where f (t, phi) = NDF at time t (h), A = degradable NDF, U = undegradable NDF, lag = time before digestion, and K = rate constant (h). A, U, and f(t, phi) are expressed as a fraction of DM at time 0. Estimates A and U did not fluctuate, whereas K and lag varied across designs. All R2 were over .96 and did not vary across designs. Comparison of designs that had the same number of observations showed that the indicator of the variance-covariance space was statistically similar across designs, although the optimal design was ranked best. Parameter estimates were similar when using different sampling schedule designs, but some estimates differed by 29%. The optimal design sampling schedule provided sufficient information to estimate parameters without loss of accuracy when compared with other designs.


A case of dermatomyositis associated with pseudomyxoma peritonei originating from mucinous adenocarcinoma of the appendix. A 69-year-old Japanese woman with erythema, severe edema on the face, Gottron's papules and poikiloderma was diagnosed as having dermatomyositis. She also noticed muscle weakness in her extremities, although her electromyogram showed neurogenic patterns. Her levels of CA19-9 and CEA were elevated and a CT of her abdomen revealed a giant, multilobular, cystic lesion in the pelvis. This tumor was diagnosed as pseudomyxoma peritonei originating from a mucinous adenocarcinoma of the appendix.


Clear cell hidradenoma: a tumor with basaliomatous changes in the overlying epidermis and follicular infundibula of surrounding skin. We report a clear cell hidradenoma on the cheek of a Japanese man. We performed the primary operation on the flesh-colored tumor, which had surface telangiectasia. The histopathologic features of the tumor, which indicated an intradermal nodular hidradenoma, consisted mainly of typical clear cells with small numbers of eosinophilic fusiform cells. Most clear cells reacted negatively for CEA, EMA, S-100 protein and KL-1 keratin, but those in and around the cystic and ductal structures reacted positively for CEA, EMA and KL-1 keratin. Ultrastructurally, these clear cells had numerous microvillus processes, abundant intracytoplasmic glycogen granules, and numerous mitochondria. In addition, buds of hyperpigmented keratinocytes hung from the overlying epidermis and thin cords of hyperpigmented keratinocytes proliferated around the follicular infundibula beside the tumor. Within two months of the primary operation, growth of the tumor into the overlying epidermis recurred rapidly. We discuss the histological features of the combination of clear cell hidradenoma with basaliomatous changes of the overlying and surrounding skin and the highly aggressive recurrence of this type of tumor after primary treatment.


Inhibitory effect of the leukotriene B4 receptor antagonist against hypomagnesic diet-induced dermatitis in hairless rats. In vivo experiments have shown that magnesium deficiency elicits characteristic skin lesions in the hairless rat. However, the mechanism of the involvement is not clarified. From the results of previous studies, a product derived from arachidonic acid, but not via the cyclooxygenase pathway, has been considered as an etiological cause. In this study, the inhibitory effect of newly produced leukotriene B4 (LTB4) receptor antagonist (LTBRA) against these skin manifestation was examined in hairless mutants given a hypomagnesic diet. Control animals with treatment of LTBRA developed eruptions on their bodies with increasing serum levels of LTB4, but LTBRA-treated animals did not suffer from the cutaneous disorder. Increased serum levels of LTB4 in control rats were significantly higher than those in the pre-treated condition. The diminution of skin lesions by LTBRA strongly indicated that magnesium deficiency dermatitis may involve a lipoxygenase-mediated metabolite of arachidonic acid, presumably LTB4, and that the cutaneous changes in hairless rats fed with hypomagnesic diet might provide an easily identifiable indicator of LTB4-mediated dermatitis.


Proteus syndrome: report of the first Japanese case with special reference to differentiation from Klippel-Trenaunay-Weber syndrome. This is the first report of a Japanese girl with Proteus syndrome. She presented with growth acceleration and precocious development of the left breast as well as macrodactyly, hemihypertrophy, a subcutaneous preaxillary mass, portwine stains, connective tissue nevi, and a depigmented macule. All these abnormalities were confined to the left side of her body. Although most of the manifestations fit those of Proteus syndrome, the presence of the portwine stains and hemihypertrophy also suggested Klippel-Trenaunay-Weber syndrome. The findings in our patient suggest that the most important characteristic distinguishing Proteus syndrome from Klippel-Trenaunay-Weber syndrome is the presence of functional abnormalities such as a growth spurt and precocious breast development. Proteus syndrome may be genetically different from the Klippel-Trenaunay-Weber syndrome.


Two cases of Fabry's disease: a hemizygote with a point mutation in the alpha-galactosidase A gene and his relative. A 34-year-old Japanese male had leg pain, edema of the legs, hypohidrosis, whorl-like opacities of the bilateral cornea, bilateral subcapsular cataracts, and chest discomfort on exercise. He had no characteristic angiokeratomas but did have telangiectases. The electrocardiogram revealed high voltage. The echocardiogram revealed mild mitral regurgitation. The alpha-galactosidase A activity in cultured lymphoblasts was deficient (0.5 nmol/h/mg protein). Electron microscopic examination of the skin revealed lamellar cytoplasmic inclusions in the endothelial cells, pericytes, and fibroblasts. He had a G--> A transition at nucleotide 982 in the coding sequence of the alpha-galactosidase A gene which resulted in a glycine to arginine amino acid substitution at residue 328. His uncle also had leg pain, edema of the legs, hypohidrosis, and chest pain on exercise. He had no characteristic angiokeratomas but did have telangiectases. Cardiovascular examination revealed hypertrophic cardiomyopathy and stenoses of coronary arteries. Electron microscopic examination of the skin revealed lamellar cytoplasmic inclusions in the endothelial cells, pericytes, and fibroblasts.


Mental health, race and ethnicity: a retrospective study of the care of ethnic minorities and whites in a psychiatric unit. A retrospective case-note study of 152 discharged psychiatric patients (77 subjects of ethnic minority origin and 75 whites) examined for details of follow-up arrangements on discharge, compliance rate with out-patient appointments and details on length of stay, status on admission, employment status, record of illicit drug taking and diagnosis. The results show that in the areas of diagnosis, possible illicit drug involvement, status on admission and length of stay, there are significant differences between Afro-Caribbean subjects and others.


IgE regulation and lymphokine patterns in aging humans. IgE production declines with age, and allergic symptoms tend to improve. Aging therefore represents an in vivo model to study IgE regulation. We compared IgE production in older (> or = 60 years old) and young (15 to 30 years old) nonatopic individuals. Addition of exogenous interleukin-4 (IL-4) to mononuclear cells from older and young subjects induced equivalent amounts of IgE, indicating that IL-4 responsiveness is preserved in aging. After surface receptor stimulation with concanavalin A, IL-4 production by mononuclear cells from older subjects was approximately 50% as compared with the young, whereas interferon-gamma (IFN-gamma) production was reduced threefold (p = 0.008). By contrast, stimulation with phorbol esters and ionophore, which bypass surface receptor signaling, induced comparable amounts of IL-4 and IFN-gamma in older and young subjects. These data point to an impairment in T-cell membrane signal transduction in older individuals. This hypothesis was directly confirmed by showing that Ca+2 fluxes after CD3 crosslinking were significantly (p = 0.014) decreased in the older population. Our findings altogether suggest that an age-dependent T-cell activation defect may result in decreased availability of IL-4 and in the waning of IgE responses.


Arachidonic acid metabolism in monocytes of aspirin-sensitive asthmatic patients before and after oral aspirin challenge. Aspirin and nonsteroidal antiinflammatory drugs induce bronchospastic reactions in patients with aspirin-sensitive respiratory disease. Although the mechanism of this reaction is unknown, all drugs that induce the respiratory reaction also inhibit the cyclooxygenase enzyme. The ensuing changes in arachidonate metabolism are presumed to play a role in the pathogenesis of the reaction. We measured generation of leukotrienes and thromboxane by calcium ionophore stimulated blood monocytes. Before aspirin challenge, monocytes released significantly more thromboxane B2 in patients with aspirin sensitivity than in patients without aspirin sensitivity or in healthy control subjects (p < 0.02). During aspirin-induced bronchospasm, release of leukotriene B4 increased significantly (45.5%, p = 0.018), whereas release of thromboxane B2 decreased (-46.9%, p = 0.028). Two hours after ingestion of 60 mg aspirin, normal monocyte release of thromboxane B2 did not drop, whereas leukotriene B4 release increased. Monocytes formed only minimal amounts of leukotriene C4. We conclude that the profile of released eicosanoids from aspirin-sensitive monocytes is distinct from non-aspirin-sensitive subjects, and that these differences could contribute to the development of bronchospasm after aspirin ingestion.