ABSTRACT
Acute renal failure can be caused by calcineurin inhibitors (CNIs), due to arteriolopathy and altered tubular function. Within this context, we present the case of a 14-month-old liver transplant recipient who suffered an acute polyuric renal failure during a short episode of hypercaloric feeding. In our case, CNI-induced distal RTA led to nephrocalcinosis and therefore to secondary nephrogenic diabetes insipidus. The diet with high renal solute load consequently resulted in an acute polyuric renal failure with severe hypernatremic dehydration. In conclusion, a hypercaloric diet in children with potentially impaired renal function due to therapy with CNIs requires precise calculation of the potential renal solute load and the associated fluid requirements.
ABSTRACT
BACKGROUND: Despite hypoglycemia and hyperglycemia being frequently observed in the early postoperative phase, information on glucose metabolism after pediatric liver transplantation (pLT) is scarce. METHODS: The goal of this retrospective single-center study, which included 46 patients who consecutively underwent 55 liver transplantations, was to gather data on glucose uptake, the prognostic relevance of hyperglycemia, and the safety of insulin administration in patients after pLT. RESULTS: In this study population, glucose intake to keep blood sugar levels (BSLs) within the targeted range of 120 to 200 mg/dL (6.7-11.1 mmol/L) increased rapidly over the first few postoperative days and was significantly correlated with graft function. There was no association between a postoperative daily mean BSL >200 mg/dL and specific posttransplant complications (acute rejection, infection, need for retransplantation, and/or death). High postoperative mean 7-day BSLs were associated with poor glucose metabolism and an increase in morbidity and 6-month posttransplant mortality. Hypoglycemia was not observed under insulin administration. CONCLUSIONS: With high BSLs being associated with poor glucose metabolism, it is likely that the critical illness itself, in addition to poor graft function, causes the increase in morbidity and mortality, with hyperglycemia serving as a marker.
Subject(s)
Blood Glucose/metabolism , Hyperglycemia/complications , Liver Transplantation , Child , Critical Illness , Female , Humans , Hyperglycemia/drug therapy , Infant , Insulin/administration & dosage , Liver Transplantation/mortality , Male , Postoperative Period , Retrospective StudiesABSTRACT
In the early phase after pediatric liver transplantation (pLT) several concomitant factors may reduce the performance of established sepsis markers. To date, their clinical interpretation is hindered by a lack of information on their postoperative kinetics. To gather more information on the postoperative course and their changes in bacterial sepsis, we prospectively studied C-reactive protein (CRP), interleukin-6 (IL-6), and procalcitonin (PCT) on 9 perioperative days in 25 consecutive pLTs. After an initial postoperative peak, IL-6 and CRP levels significantly re-increased in patients with bacterial sepsis (P < .001). In contrast, PCT had very high postoperative levels; therefore severe infection was a comparatively inferior trigger for PCT elevation compared with the initial operation. The area under the receiver operating characteristic curve to diagnose postoperative sepsis for PCT was only 0.52, compared with 0.95 for IL-6 and 0.89 for CRP. None of the studied biomarkers were depressed by poor graft function. In conclusion, PCT performs poorly as a biomarker for sepsis in the early phase after pLT. With a rapid decline of initially elevated levels, IL-6 provides the best kinetics for detection of postoperative bacterial sepsis.
Subject(s)
C-Reactive Protein/metabolism , Calcitonin/blood , Interleukin-6/blood , Liver Transplantation , Postoperative Complications , Protein Precursors/blood , Sepsis/blood , Adolescent , Biomarkers/blood , Calcitonin Gene-Related Peptide , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Prospective Studies , ROC Curve , Sepsis/etiologyABSTRACT
Minimally invasive telerobotic surgical systems enable surgeons to perform complicated procedures without large incisions. Unfortunately, these systems typically do not provide the surgeon with sensory feedback aside from stereoscopic vision. We have, thus, developed VerroTouch, a sensing and actuating device that can be added to Intuitive Surgical's existing da Vinci S Surgical System to provide auditory and vibrotactile feedback of tool contact accelerations. These cues let the surgeon feel and hear contact with rough textures as well as the making and breaking of contact with objects and other tools. To evaluate the merits of this approach, we had 11 surgeons use an augmented da Vinci S to perform three in vitro manipulation tasks under four different feedback conditions: with no acceleration feedback, with audio feedback, with haptic feedback, and with both audio and haptic. Subjects expressed a significant preference for the inclusion of tool contact acceleration feedback, although they disagreed over which sensory modality was best. Other survey responses and qualitative written comments indicate that the feedback may have improved the subject's concentration and situational awareness by strengthening the connection between the surgeon and the surgical instruments. Analysis of quantitative task metrics shows that the feedback neither improves nor impedes the performance of the chosen tasks.
ABSTRACT
The tibial plateau levelling osteotomy (TPLO) is commonly performed for treatment of cranial cruciate ligament deficiency in dogs. In order to be performed as described, this procedure relies on consistent measurement of the tibial plateau angle (TPA) on radiographs. This prospective study compared two radiographic methods for subsequent TPA measurement with respect to measured angle and ease of determining landmarks for measurement as determined by four observers. One method was the accepted standard radiographic protocol outlined in the TPLO training seminars. The other method involved a novel split image radiographic protocol not yet described in the literature. Participants' subjective scores as to ease of identifying landmarks and determining TPA on radiographs for each method were evaluated. Inter-observer TPA measurement variability was also assessed for each method. The novel radiographic method was judged to be significantly better in terms of ease of measuring TPA. Inter-observer measurement variability was considered appropriate for recommending use of this novel method for radiographing patients for TPA measurements.
Subject(s)
Anterior Cruciate Ligament , Dog Diseases/diagnostic imaging , Joint Diseases/veterinary , Stifle/diagnostic imaging , Tibia/diagnostic imaging , Animals , Dog Diseases/surgery , Dogs , Female , Joint Diseases/diagnostic imaging , Male , Observer Variation , Prospective Studies , Radiography/veterinaryABSTRACT
A 27-year-old man was admitted with high fever and shivers eleven days after returning from vacation in Indonesia. Physical examination, laboratory values, abdominal ultrasound, and chest x-ray were not conclusive. All blood cultures yielded growth of Salmonella enterica serovar Typhi, and typhoid fever was diagnosed. Subsequently, the patient developed septic shock and pulmonary edema. In this case report epidemiological, clinical, and therapeutic aspects of typhoid fever are discussed with special emphasis on criteria for severe typhoid fever, which is treated with additional glucocorticoids.
Subject(s)
Dexamethasone/therapeutic use , Fever of Unknown Origin/etiology , Shock, Septic/etiology , Travel , Typhoid Fever/complications , Typhoid Fever/drug therapy , Adult , Anti-Inflammatory Agents/therapeutic use , Fever of Unknown Origin/diagnosis , Humans , Indonesia , Male , Shock, Septic/diagnosis , Typhoid Fever/diagnosisABSTRACT
The current study tests the hypothesis that basal level and minute-by-minute correction of plasma Ca2+ by outward and inward Ca2+ fluxes from and into an exchangeable ionic pool in bone is controlled by an active partition system without contributions from the bone remodeling system. Direct real-time measurements of Ca2+ fluxes were made using the scanning ion-selective electrode technique (SIET) on living bones maintained ex vivo in physiological conditions. SIET three-dimensional measurements of the local Ca2+ concentration gradient (10 microm spatial resolution) were performed on metatarsal bones of weanling mice after drilling a 100-mum hole through the cortex to expose the internal bone extracellular fluid (BECF) to the bathing solution, whose composition mimicked the extracellular fluid (ECF). Influxes of Ca2+ towards the center of the cortical hole (15.1+/-4.2 pmol cm-2 s-1) were found in the ECF and were reversed to effluxes (7.4+/-2.9 pmol cm-2 s-1) when calcium was depleted from the ECF, mimicking a plasma demand. The reversal from influx to efflux and vice versa was immediate and fluxes in both directions were steady throughout the experimental time (>or=2 h, n=14). Only the efflux was nullified within 10 min by the addition of 10 mM/L Na-Cyanide (n=7), demonstrating its cell dependence. The timeframes of the exchanges and the stability of the Ca2+ fluxes over time suggest the existence of an exchangeable calcium pool in bone. The calcium efflux dependency on viable cells suggests that an active partition system might play a central role in the short-term error correction of plasma calcium without the contribution of bone remodeling.
Subject(s)
Bone Resorption , Bone and Bones/metabolism , Calcium/metabolism , Animals , Ion Transport , Mice , Mice, Inbred BALB CABSTRACT
Propofol is used for the treatment of refractory status epilepticus. When given as a long-term infusion propofol may cause a rare but frequently fatal complication, the propofol infusion syndrome. The hallmarks are metabolic acidosis, lipemia, rhabdomyolysis and myocardial failure. Propofol infusion syndrome is caused by impaired fatty acid oxidation. Beside anticonvulsants the ketogenic diet, a high-fat, low-carbohydrate, adequate-protein diet, is an effective treatment for difficult-to-control seizures. We report a 10-year-old boy with catastrophic epilepsy, who developed fatal propofol infusion syndrome when a ketogenic diet was initiated. Substances like propofol which impair fatty acid oxidation may pose an increased risk if combined with ketogenic diet.
Subject(s)
Anticonvulsants/adverse effects , Propofol/adverse effects , Status Epilepticus/diet therapy , Status Epilepticus/drug therapy , Tachycardia, Ventricular/etiology , Acidosis/etiology , Anticonvulsants/administration & dosage , Child , Diet Therapy/adverse effects , Fatal Outcome , Humans , Infusions, Parenteral , Male , Propofol/administration & dosage , Rhabdomyolysis/etiology , SyndromeABSTRACT
An animal-vegetal net ionic current identified previously using voltage probe techniques in maturing Xenopus laevis oocytes has now been investigated using noninvasive ion-selective microelectrodes. Three-dimensional fluxes of hydrogen (H(+)), potassium (K(+)), and bicarbonate (HCO(3)(-)) were characterized with respect to the developmental stage and hemisphere of the oocyte and presence of surrounding follicular tissue. Variable effluxes of H(+) and HCO(3)(-) were recorded from both the animal and vegetal hemispheres. Variable influxes and effluxes of K(+) were also observed. The equatorial region, silent by voltage probe, exhibited fluxes of H(+) and K(+). Simultaneous measurement of pairs of ions allowed correlation analysis of two ion types. Notably for H(+) and K(+) data, positive and negative correlation at animal and vegetal poles respectively offer an explanation of the unpredictable results obtained when individual ions were observed independently.
Subject(s)
Bicarbonates/pharmacokinetics , Ion Channels/physiology , Oocytes/physiology , Potassium/pharmacokinetics , Xenopus laevis/physiology , Animals , Electrophysiology , Embryo, Nonmammalian/physiology , Female , Potassium/chemistry , Protons , Xenopus laevis/embryologyABSTRACT
The occurrence of oscillatory behaviours in living cells can be viewed as a visible consequence of stable, regulatory homeostatic cycles. Therefore, they may be used as experimental windows on the underlying physiological mechanisms. Recent studies show that growing pollen tubes are an excellent biological model for these purposes. They unite experimental simplicity with clear oscillatory patterns of both structural and temporal features, most being measurable during real-time in live cells. There is evidence that these cellular oscillators involve an integrated input of plasma membrane ion fluxes, and a cytosolic choreography of protons, calcium and, most likely, potassium and chloride. In turn, these can create positive feedback regulation loops that are able to generate and self-sustain a number of spatial and temporal patterns. Other features, including cell wall assembly and rheology, turgor, and the cytoskeleton, play important roles and are targets or modulators of ion dynamics. Many of these features have similarities with other cell types, notably with apical-growing cells. Pollen tubes may thus serve as a powerful model for exploring the basis of cell growth and morphogenesis. BioEssays 23:86-94, 2001.
Subject(s)
Pollen/growth & development , Animals , Calcium/metabolism , Cytosol/metabolism , Pollen/metabolism , ProtonsABSTRACT
The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-free continuous culture at steady-state dissolved oxygen (DO) concentrations of 10%, 50%, and 100% of air saturation in both LH Series 210 (LH) and New Brunswick Scientific (NBS) CelliGen bioreactors. All culture parameters were monitored and controlled and were nominally identical at steady state in the two bioreactors. The secreted monoclonal antibody (mAb), an immunoglobulin G(1), was purified and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGase F). Asparagine-linked (N-linked) oligosaccharide pools released from mAb samples cultured in each bioreactor at each of the three DO setpoints were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialyl oligosaccharides and trace amounts of afucosyl oligosaccharides. The level of DO affects the glycosylation of this mAb. A definite reduction in the level of galactosylation of N-glycan chains was observed at lower DO in both bioreactors, as evidenced by prominent increases in the relative amounts of agalactosyl chains and decreases in the relative amounts of digalactosyl chains-with the relative amounts of monogalactosyl chains being comparatively constant. However, the quantitative results are not precise matches between the two bioreactors. The effect of DO on galactosylation is less pronounced in the NBS bioreactor than in the LH bioreactor, particularly the shift between the relative amounts of agalactosyl and digalactosyl chains in 10% and 50% DO. There are also perceptibly higher levels of sialylation of the mAb glycans in the NBS bioreactor than in the LH bioreactor at all three DO setpoints. The results indicate that the DO effect is not bioreactor specific and that nominally identical steady-state conditions in different chemostat bioreactors may still lead to some incongruities in glycosylation, possibly due to the particular architectures of the bioreactors and the design of their respective monitoring and control systems. The observed differences in N-linked glycosylation of the mAb secreted by the hybridoma grown in the LH and NBS bioreactors may be explained by the differences in oxygen supply and control strategies between the two bioreactors.
Subject(s)
Antibodies, Monoclonal/metabolism , Bioreactors , Amidohydrolases/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Carbohydrate Sequence , Cell Culture Techniques , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Glycosylation , Hybridomas/immunology , Mice , Molecular Sequence Data , Oligosaccharides/chemistry , Oligosaccharides/isolation & purification , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine AmidaseABSTRACT
BACKGROUND: The staphylococcal protein A (SPA) column used to treat refractory autoimmune and alloimmune thrombocytopenia and rheumatoid arthritis patients is primed with heparin to prevent possible fibrin clot formation when the patient's plasma is passed through the column. A BMT patient with refractory alloimmune thrombocytopenia had prolonged activated partial thromboplastin times (aPTTs) at the end of SPA column treatments. This observation led to in vivo and in vitro analysis of the kinetics of heparin elution from the SPA column. STUDY DESIGN AND METHODS: Two patients with refractory rheumatoid arthritis, who were treated on five occasions with the SPA column (as a part of a national trial) primed with 5000 U of heparin, were monitored for aPTT and heparin in their plasma. In addition, two in vitro analyses were performed with FFP for heparin elution from the SPA column. RESULTS: The in vivo studies showed the presence of 0.3 to 1.5 U per mL of heparin in patients' plasma at the end of the SPA column treatments that corresponded with the prolonged aPTTs. The in vitro studies showed that 82 to 85 percent heparin (approx. 4400 U) was eluted from the SPA column during rather than before the procedure. CONCLUSION: Patients undergoing SPA column treatments, especially those with thrombocytopenia, may be at increased risk of bleeding as a result of the presence of a significant amount of heparin in their circulation during the entire period of SPA column treatment.
Subject(s)
Anticoagulants/adverse effects , Arthritis, Rheumatoid/therapy , Autoimmune Diseases/therapy , Chromatography, Affinity , Hemorrhagic Disorders/chemically induced , Heparin/adverse effects , Immunosorbent Techniques , Staphylococcal Protein A , Thrombocytopenia/therapy , Anticoagulants/blood , Antigen-Antibody Complex/isolation & purification , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/complications , Autoimmune Diseases/blood , Autoimmune Diseases/complications , Autoimmune Diseases/immunology , Hemorrhagic Disorders/etiology , Heparin/blood , Humans , Immunoglobulin G/isolation & purification , Isoantibodies/immunology , Partial Thromboplastin Time , Thrombocytopenia/blood , Thrombocytopenia/complications , Thrombocytopenia/immunologyABSTRACT
Nursing and computers will enter the new millennium in synergy. As a science, nursing is using computers to organize and communicate information to expedite the nursing process and provide safe patient care. In 1992, the American Nursing Association recognized a new specialty in nursing: Nursing Informatics, a specialty that will provide the ability to adapt to the ever-changing future technology.
Subject(s)
Career Mobility , Computers , Perioperative Nursing/trends , HumansABSTRACT
The developmental fate of the vitellogenin-derived yolk protein, lipovitellin (Lv), was investigated in winter flounder embryos and yolk-sac larvae. Since Lv is present as only one major polypeptide in ovulated winter flounder eggs, unlike the multiple yolk polypeptides found in the mature eggs of most teleosts, this system is presented as a simpler model of yolk protein structure and utilization during teleostean development. Winter flounder Lv is cleaved during embryogenesis from a 94 kD polypeptide at fertilization to 67 kD and 26 kD polypeptides at hatching. The rate of this proteolytic processing is slow during early embryonic development, but enters a more rapid phase between days 8 and 12 post-fertilization in embryos reared at 4-5 degrees C, and approaches 50% completion at day 10. Lv processing is essentially complete 3 days before hatching; nevertheless, major degradation of the Lv peptide by the developing winter flounder does not occur until after hatching. The Stokes radius of Lv changes only moderately following processing, from 4.50 nm in unfertilized eggs to 4.19 nm in late embryos and newly hatched larvae, whereas the processed Lv retains its heat stability relative to other yolk polypeptides. Nearly 50% of its lipid content, however, is released from the Lv particle during embryogenesis, concomitant with cleavage of the Lv 94 kD polypeptide. Lv processing may thus render a portion of the yolk protein-associated lipid more accessible to the developing embryo, whereas other yolk components are retained for later use by the winter flounder larva. Alternately, removal of lipid may lead to proteolytic vulnerability of the Lv polypeptide. In either case, only a portion of the lipid moiety of the Lv particle appears to play a significant nutritive role for the embryo, whereas its protein component is reserved for larval use. J. Exp. Zool. 284:686-695, 1999.
Subject(s)
Egg Proteins, Dietary/metabolism , Embryo, Nonmammalian/metabolism , Flounder/physiology , Larva/metabolism , Animals , Cell Extracts/analysis , Chromatography, Gel , Egg Proteins , Egg Proteins, Dietary/analysis , Egg Proteins, Dietary/isolation & purification , Electrophoresis, Polyacrylamide Gel , Embryo, Nonmammalian/embryology , Female , Hot Temperature , Larva/growth & development , Lipids/analysis , Male , Oocytes/chemistryABSTRACT
Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H+) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 microM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.
Subject(s)
Pollen/growth & development , Pollen/metabolism , Diffusion , Fluoresceins , Fluorescent Dyes , Hydrogen-Ion Concentration , Microscopy, Fluorescence , Models, Biological , Pollen/ultrastructureABSTRACT
The murine B-lymphocyte hybridoma, CC9C10, was grown at steady state in serum-free continuous culture at dissolved oxygen (DO) concentrations of 10, 50, and 100% of air saturation. The secreted mAb, an IgG1, was purified and subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Both methods resulted in complete removal of N-linked oligosaccharide chains. Isolated N-glycan pools were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The FACE profiles and corresponding HPAEC-PAD chromatograms of N-linked oligosaccharides obtained by PNGase F digestion and hydrazinolysis provided complementary and corroborating information. The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialylated, and trace amounts of afucosyl, oligosaccharides. A definite shift towards decreased galactosylation of glycan chains was observed as DO concentration in continuous culture was reduced. The vast majority of N-linked glycosylation occurred on the heavy chain. There was no evidence for N-linked glycosylation of the light chain or for O-linked glycosylation of the mAb.
Subject(s)
B-Lymphocytes/metabolism , Hybridomas/metabolism , Oxygen/pharmacology , Animals , Antibodies, Monoclonal/chemistry , Carbohydrate Sequence , Cell Culture Techniques/methods , Electrophoresis, Polyacrylamide Gel , Glycoside Hydrolases/metabolism , Glycosylation/drug effects , Immunoglobulin G/chemistry , Mice , Molecular Sequence Data , Oligosaccharides/chemistry , Polysaccharides/chemistryABSTRACT
Hair samples of 20 volunteers of the techno-music scene, who more or less regularly consumed ecstasy tablets and speed and anonymously reported their abuse history, were analyzed in one to seven 3 cm segments for amphetamine (A), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethamphetamine (MDE) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butylamine (MBDB) by digestion in 1 M NaOH, subsequent extraction with C18 Bond Elut columns, derivatization with pentafluoropropionyl anhydride and GC/MS-SIM measurements using deuterated standards of A, MA, MDA and MDMA. The concentrations were in the regions 0.1 to 4.8 ng/mg for A (17 samples), 0.05 to 0.89 ng/mg for MDA (16 samples), 0.1 to 8.3 ng/mg for MDMA (16 samples), 0.12 to 15 ng/mg for MDE (13 samples) and 0.21 to 1.3 ng/mg for MBDB (2 samples). MA was not detected. For comparison the frequency and the concentration of these drugs in 124 different ecstasy tablets were determined by HPLC. The drug concentration in the hair segments were compared with the volunteers' reports. Despite the enormous interindividual differences qualitatively an increase of the total concentration of MDA, MDMA and MDE in the proximate 3 cm segments with increasing ecstasy abuse frequency during the last three month before sampling is recognized. In the individual comparison with the chronological consumer reports in most cases a longer interruption or a change of the abuse intensity is not clearly seen at the segment concentrations. As a reason the incorporation of the drugs from sweat into elder hair regions and the slow removal by washing are discussed.
Subject(s)
Amphetamine/analysis , Chromatography, High Pressure Liquid/methods , Gas Chromatography-Mass Spectrometry/methods , Hair/chemistry , Hallucinogens/analysis , N-Methyl-3,4-methylenedioxyamphetamine/analysis , Substance Abuse Detection/methods , Substance-Related Disorders/diagnosis , Adolescent , Adult , Amphetamine/chemistry , Baths , Female , Hair Color , Hallucinogens/chemistry , Humans , Male , N-Methyl-3,4-methylenedioxyamphetamine/chemistry , Time FactorsABSTRACT
An enzyme-linked immunoabsorbent assay was developed for detection and quantification of the yolk protein lipovitellin (Lv) and its plasma precursor, vitellogenin (Vg), in winter flounder (Pleuronectes americanus). Native Lv was found to be a mixture of heat-stable and heat-labile molecules in mature, ovulated eggs. A heat-stable Lv fraction was purified from extracts of unfertilized eggs by brief heat treatment and gel permeation chromatography on Bio-Gel A-1.5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of heat-stable Lv revealed a single polypeptide of 94 kD, while native Lv also possessed several smaller polypeptides, suggesting that heat-labile Lv contains proteolytic cleavages of the 94-kD polypeptide which destabilize its structure. The Stokes radius of the native protein on Bio-Gel A-1.5 was estimated at 4.50 nm, while the Stokes radii of heat-stable and heat-labile Lv were 4.26 nm and 5.17 nm, respectively. Heat-stable Lv was used to produce a rabbit polyclonal antiserum which reacted with a single 175-kD polypeptide in Western blots of vitellogenic female winter flounder serum, but did not react with any component of male serum. Ouchterlony double diffusion using this antiserum demonstrated immunological identity of Lv, heat-stable Lv, and Vg. The anti-Lv anti-serum was used to construct an homologous ELISA with a linear response between 25 and 300 ng/ml. This assay was used to characterize a Bio-Gel A-1.5 column profile of serum from an estradiol-treated male winter flounder, and a single peak, with Stokes radius of 6.70 nm, was identified as Vg. Winter flounder Vg was confirmed to be a dimer, while Lv from mature eggs was found to be a monomer of a lower molecular weight polypeptide.
Subject(s)
Egg Proteins/chemistry , Egg Yolk/chemistry , Flounder , Immunoassay/methods , Ovum/chemistry , Vitellogenins/analysis , Animals , Antibody Specificity , Egg Proteins/immunology , Egg Yolk/immunology , Enzyme-Linked Immunosorbent Assay , Female , Immunodiffusion , Male , Ovum/immunology , Vitellogenins/immunologyABSTRACT
The vagina and medulla of the adrenal gland of mice vaginally infected with herpes simplex virus (HSV) types 1 and 2 were examined in the latent stage of infection (5 to 51 weeks post-infection). RNA in situ hybridization with HSV-1 and -2 latency-associated transcript (LAT) RNA probes resulted in positively stained neuronal cell nuclei in the uterovaginal plexus, but not in the medulla of the adrenal gland. These organs were chosen because HSV antigens can be detected not only in the vaginal epithelium, but also in neurons of the uterovaginal plexus and in the medulla of the adrenal gland at the acute stage of genital infection. To our knowledge, this is the first report describing LATs in neurons of the uterovaginal plexus in the genital tract of latently HSV-infected mice.