Radioimmunoassay of arginine vasopressin in the plasma and urine.

We introduced the radioimmunoassay (RIA) of arginine vasopressin (AVP) with standard AVP and antiserum to AVP (both Calibiochem). The sensitivity of the system was increased from the declared 4pg to 1 pg per tube by preparing AVP-125I of high specific activity (about 1,500 mCi/mg) and by modifying the reaction conditions. The sensitivity of the method was adequate for measuring AVP in urine and in concentrated plasma extracts, even under physiological conditions. Reliability of the results depended upon maintenance of approximately the same osmolarity in all the RIA samples. The mean plasma AVP level, uncorrected for AVP extraction losses, was 1.52 +/- 0.20 pg/ml for an ad libitum fluid intake; in fluid deprivation it rose in proportion to the osmolarity of the plasma to 5.83 +/- 0.42 pg/ml at 12 hours and to 19.09 +/- 4.51 pg/ml at 36 hours. Extraction recovery of added AVP was about 63%. The urinary AVP concentration varied according to the patients' state of hydratation from undetectable values at UOsm less than 200 mOsm/1 to a mean 16.5 +/- 7.9 pg/ml in the presence of an ad libitum fluid intake and to 29.1 +/- 7.5 pg/ml after 12 hours' and 117.2 +/- 13.7 pg/ml after 36 hours' deprivation of fluids.     

Changes in the titer of the female-predominant storage protein (81K) during larval and pupal development of the waxmoth,galleria mellonella

The levels of an 81K storage protein in the waxmoth, Galleria mellonella, were monitored during the course of development using rocket immunoelectrophoresis. During the fifth and sixth larval stadia, 81K protein levels increased during feeding and growth but sharply declined at each larval molt. During the fifth and sixth stadia hemolymph levels of the 81K protein increased to about 1 and 2.5 mg/ml, respectively, with no discernible differences between levels in males and females. Neither the fat body nor the remainder of the carcass contained the 81K protein, indicating that the accumulation of this protein during the intermolt period was exclusively in the hemolymph and redistribution of the 81K protein into other tissues does not occur at the final two larval molts. During the seventh (final) larval stadium the absolute quantities of the 81K protein increased from 23 μg per insect to over 1,600 μg in females and to 300 μg in males. The hemolymph concentration of the 81K protein reached 28 mg/ml in females and 6 mg/ml in males with only low levels found in the remaining tissues. Shortly after pupal apolysis, marked by eyespot retraction, the fat body in both sexes rapidly and quantitatively sequestered the 81K protein from the hemolymph. The 81K protein in the hemolymph of both males and females rapidly dropped to nearly zero concentration by pupation. The 81K storage protein remained localized in the fat body cells after uptake occurred, even though the fat body cells disaggregate and reaggregate during metamorphosis. During pharate adult development the 81K storage protein disappeared from the fat body without entering the hemolymph. At adult eclosion 81K was virtually absent from the tissues of both males and females.     

Development of cerebrospinal fluid and the blood-cerebrospinal fluid barrier in rabbits.

Blood plasma and cerebrospinal fluid (CSF) samples were collected from adult female rabbits (New Zealand White), newborn, and embryos at 18, 20, 24, and 28 days of gestation. Samples were analyzed for total protein using the Folin phenol reagent. During development, mean total protein of blood plasma rose sharply from 12.45 to 12.51 mg/ml at 18 to 20 days to 37.56 mg/ml at 28 days. Levels further increased to 54.06 mg/ml in the newborn and to 66.18 mg/ml in the adult. The protein concentration of cerebrospinal fluid was constant at 5.20 to 5.29 mg/ml between 18 and 20 days of gestation, but steadily decreased to 3.53 mg/ml at 28 days. By birth, the CSF protein concentration was further reduced to 2.08 mg/ml, and this level differed only slightly (P < 0.05) from CSF protein values determined for adults. These data indicate that the blood-cerebrospinal fluid barrier to proteins begins to function by 18 to 20 days of gestation, and the protein concentration of cerebrospinal fluid approaches the normal adult value soon after birth.     

DIFFERENTIATION OF MURINE MARROW MEGAKARYOCYTE PROGENITORS (CFUM): HUMORAL CONTROL IN VITRO

Differentiation of mouse marrow megakaryocyte progenitors (CFUm) was studied in vitro by a colony assay using a plasma clot system. Erythropoietin (EPO) from sheep plasma (6 units/mg protein) in doses from 1 to 5 units/ml induced a linear increase in CFUm to a maximum of 20 colonies/10⁵ cells plated. Human urinary EPO also induced a dose-responsive increase in CFUm, but the maximum was 9 colonies/10⁵ with 2·0 units/ml of EPO and there was a decrease in colonies above that concentration. Thrombocytopoiesis-stimulating factor (TSF) derived from human embryonic kidney culture supernatant fluids induced a dose-responsive increase in CFUm in concentrations from 0·01 to 0·32 mg protein/ml in the absence of added EPO. TSF did not support the growth in vitro of erythroid colonies from mouse marrow (CFUe and BFUe) indicating an absence of EPO activity. In these studies sheep EPO appeared more effective in supporting CFUe growth than human EPO. TSF also had a stimulatory function in megakaryocyte differentiation at a precursor level. Multiple humoral factors play a role in megakaryocytopoiesis in vitro.     

Elevated Nerve Growth Factor Levels in the Synovial Fluid of Patients with Inflammatory Joint Disease

A novel pH shock extraction procedure was used to measure nerve growth factor (NGF) levels in both normal and inflamed synovial fluids using a sensitive and specific two-site enzyme linked immunosorbant assay. To date no data is available on NGF levels in normal synovial fluids. Synovial fluids were taken from 5 normal volunteers, 12 patients with rheumatoid arthritis and 10 patients with other inflammatory arthropathies. The mean ± SEM NGF concentration in normal synovial fluids was 95 ± 33.2 pg/ml (range 39.1–143.1 pg/ml), whereas the mean NGF concentration in the synovial fluids taken from patients with rheumatoid arthritis was 532.5 ± 123.8 pg/ml (range 152–1686 pg/ml). The mean NGF concentration in patients with other inflammatory arthropathies was also raised (430.6 ± 90 pg/ml; range 89–1071 pg/ml). The NGF concentrations were significantly higher in the synovial fluids from both inflamed groups (ANOVA p < 0.05) compared to normals. Raised levels of NGF in synovial fluid may contribute directly to joint inflammation via activation of inflammatory cells.     

Tissue osmolarity of the New Zealand land flatworm (Arthurdendyus triangulatus)

Tissue fluid osmolarity of flatworms kept with moist bark was 243+/-4 S.E.M. mOsm kg(-1). Tissue fluid osmolarity of those kept with water-saturated tissue paper was 205+/-5 S.E.M. mOsm kg(-1). Flatworms placed in water of 300 and 400 mOsm kg(-1) lost weight. Those placed in water of 0, 100 and 200 mOsm kg(-1) gained weight. This suggests that body tissue fluids were approximately 260 mOsm kg(-1). Tissue fluids were slightly hyperosmotic in external media of 200, 300 and 400 mOsm kg(-1), and strongly hyperosmotic at 0 and 100 mOsm kg(-1). The highest measured value of tissue osmolarity was 457 mOsm kg(-1) from a specimen in a medium of 400 mOsm kg(-1). The lowest value was 145 mOsm kg(-1) from a specimen in pure water. Transverse sections of flatworms from different media concentrations suggest that fluids are absorbed into or removed from all tissues.     

Osmotic and ionic haemolymph concentrations in the Baltic Sea amphipod Gammarus oceanicus in relation to water salinity

The aim of this work was to determine the osmotic and ionic (Na(+), K(+), Ca(2+), Mg(2+) and Cl(-)) haemolymph concentrations in Gammarus oceanicus at different salinity levels. Being a species of marine origin it inhabits brackish waters of the Baltic Sea. G. oceanicus specimens were collected in January 2003 from the Gulf of Gdansk (salinity 7 psu). The animals were gradually acclimated to eight different salinity levels (5, 7, 14, 20, 25, 30, 35 and 41 psu) at a temperature of 5 degrees C and 100% oxygen saturation. The haemolymph osmolalities correlated positively with external salinity, from 545.4+/-17.3 mOsm in 5 psu to 1185.9+/-34.6 mOsm in 41 psu. G. oceanicus hyperregulated within the 5-31.5 psu range; above 31.5 psu it hyporegulated its body fluids in comparison to the external medium. At 31.5 psu (1017 mOsm) the haemolymph concentration of G. oceanicus was isoosmotic with the habitat. The haemolymph concentrations of all the studied ions, except K(+), correlated positively with their concentrations at the various salinity.     

Elimination kinetics and protein binding of theophylline in the rabbit

The detailed elimination kinetics of theophylline were studied in 27 rabbits. Each received a 10 mg/kg intravenous bolus of aminophylline. The theophylline half-life ($\text{T}\text{1}\text{2}$) was 3.8 ± 0.63 hr. The apparent volume of distribution (V_D) and total body clearance (TBC) for theophylline were 439 ± 60 ml/kg and 81.0 ± 17.3 ml/kg·hr respectively. Theophylline protein binding was determined in 10 animals. The mean bound fraction was 74.3 ± 3.9% (range, 68.3–78.0%); the fraction bound was concentration indifferent over a serum concentration range of 5–20 μgm/ml.     

Biochemical aspects of Balanus hameri haemolymph,together with some comparative haemolymph data for Balanus balanus and Lepas anatifera

• 1.1. The concentration of protein in the haemolymph of Balanus hameri ranged from 2.0 to 17.3 mg/ml, and the lipid from 1.4 to 7.7 mg/ml; the haemolymph protein and lipid levels increased significantly prior to cross-fertilization.
• 2.2. The protein and lipid concentrations in Balanus balanus haemolymph were 8.1 and 1.7 mg/ml respectively.
• 3.3. The lipid concentration of Lepas anatifera haemolymph was 1.2 mg/ml.
• 4.4. The neutral lipid and phospholipid components of B. hameri and L. anatifera haemolymph were the same, with the major components of the phospholipid fraction being phosphatidyl ethanolamine and phosphatidyl choline.
• 5.5. The osmolarity (970.4 mOsm), chloride ion concentration (501.3 m-eq/l) and pH (7.29) of B. hameri haemolymph were also determined.

     

The chemical composition of the follicular fluid of the teleost Clinus dorsalis (Perciformes: Clinidae)

• 1.1. The osmolarity and pH of the follicular fluid was determined and analyses of total glucose, total lipids, total proteins, amino acids, urea, sodium and potassium carried out.
• 2.2. The mean osmolarity of the follicular fluid was found to be 325 mOsm/kg and the mean pH was 7.9.
• 3.3. The embryotrophe was rich in lipids (1092.39 mg/100 ml) and amino acids with the amino acid concentration exceeding normal values for human plasma.

     

Biophysical and biochemical characteristics of bactrian camel semen collected by artificial vagina

Seminal characteristics were investigated in Bactrian camel in this study. Semen samples from ten mature Bactrian camel bulls were collected using a modified bovine artificial vagina. The biophysical parameters including volume, color, sperm concentration and fast forward progressive motility, percentage of live sperm and the biochemical parameters including osmolarity, pH, glucose, calcium, phosphorus, chloride, triglycerides, phospholipids, total protein, albumin and non-protein nitrogen concentrations in seminal plasma were measured. The mean time for semen collection was 5.3 +/- 0.29 min. The volume of semen varies from 1.2 to 26 (8.2 +/- 0.7 mls). The majority of semen samples (83.6%) were milky in color and consistency. The average osmolarity of semen was 316.1 +/- 1.48 mOsm/kg H(2)O. The pH of semen was slightly alkaline (7.4 +/- 0.03). The mean concentration of spermatozoa was 414.8 +/- 25.04 x 10(6)cells/ml. The fast forward progressive motility of spermatozoa was 62.4 +/- 1.57%. The percentage of live spermatozoa was 85.6 +/- 1.15. Seminal plasma concentration of glucose was 35.8 +/- 0.9 mg/dl. Non-protein nitrogen, total protein and albumin were 32.5 +/- 2.5, 2200 +/- 100 and 1100 +/- 100mg/dl, respectively. The average concentrations of phospholipids and triglycerides in seminal plasma were 36.4 +/- 2.1 and 101.6 +/- 5.5mg/dl, respectively. The concentrations of calcium, phosphorus and chloride were 8.2 +/- 0.1, 2.9 +/- 1.7 mg/dl and 97.9 +/- 2.9 mEq./l, respectively.     

Isolated respiring heart mitochondria release reactive oxygen species in states 4 and 3

Isolated mitochondria respiring on physiological substrates, both in state 4 and 3, are reported to be or not to be a source of reactive oxygen species (ROS). The cause of these discrepancies has been investigated. As protein concentration was raised in in vitro assays at 37°C, the rate of H₂O₂ release by rat heart mitochondria supplemented with pyruvate/malate or with succinate (plus rotenone) was shown to increase (0.03–0.15?mg?protein/ml), to decrease (0.2–0.5?mg?protein/ml) and to be negligible (over 0.5?mg?protein/ml). The inhibition of mitochondrial respiration (with rotenone or antimycin A) or the increase in the oxygen concentration dissolved in the assay medium allowed an enhancement of ROS production rate throughout the studied range of protein concentrations. In mitochondria respiring in state 3 on pyruvate/malate or on succinate (plus rotenone), ROS release vanished for protein concentrations over 0.5 or 0.2?mg/ml, respectively. However, ROS production rates measured with low protein concentrations (below 0.1?mg/ml) or in oxygen-enriched media were similar or even slightly higher in the active respiratory state 3 than in the resting state 4 for both substrates. Consequently, these findings indicate that isolated mitochondria, respiring in vitro under conditions of forward electron transport, release ROS with Complex I- and II-linked substrates in the resting condition (state 4) and when energy demand is maximal (state 3), provided that there is sufficient oxygen dissolved in the medium.     

Effect of a high protein diet on glucose tolerance in the rat model

The purpose of this study was to determine the effects of a high protein diet on glucose tolerance. Nine Sprague Dawley rats received a high protein (HP) diet (65% protein, 35% fat) and eight rats consumed a standard chow (SC) diet over eight weeks. Oral glucose tolerance tests (OGTT) were performed at the end of the third and the seventh week. The diet did not effect glucose tolerance in the first (SC=10357+/-294 mg/dl/120 min; HP=9846+/-300 mg/dl/120 min) or the second OGTT (SC=10134+/-395 mg/dl/120 min; HP=10721+/-438 mg/dl/120 min) as reflected by the area under the glucose concentration curve. Similarly, the area under the insulin concentration curve was not effected by the high protein diet during the first (SC=49.21+/-8.46 ng/ml/120 min; HP=41.75+/-10.54 ng/ml/120 min) or the second OGTT (SC=96.63+/-13.68 ng/ml/120 min; HP=92.77+/-17.44 ng/ml/120 min). The high protein diet group experienced a delayed glucose response for the first (SC=30 min at 112+/-7 mg/dl; HP=60 min at 101+/-5 mg/dl) and second OGTT (SC=15 min at 117+/-5 mg/dl; HP=60 min at 95+/-7 mg/dl). Body mass increased to the same extent in each diet group from the initial to final weighing (SC=159+/-2 g to 254+/-7 g; HP=157+/-2 g to 242+/-7 g). Despite a delay in peak glucose response, these findings suggest that glucose tolerance and body mass were neither adversely nor positively affected by a high protein diet.     

Enzyme-Linked Immunosorbent Assay for the Human Glial Fibrillary Acidic Protein Using a Mouse Monoclonal Antibody

Two enzyme-linked immunosorbent assays (ELISAs) have been developed for the quantification of soluble human glial fibrillary acidic protein (GFAP). The specificity of the assays for GFAP is ensured by the use of a monoclonal antibody directed against a GFAP-specific antigenic determinant. One ELISA is a four-layer system working in the concentration range 5-600 ng GFAP/ml. The other ELISA is a five-layer system and includes a biotin/avidin binding reaction. The latter assay has a working range of 0.5-60 ng GFAP/ml. The assays may be used for quantification of GFAP in CSFs, amniotic fluids, and extracts or homogenates of normal and pathological brain material. GFAP in serum could not be quantified because of unidentified interference. CSFs from 18 nonneurological subjects were found to contain 2-14 ng GFAP/ml (mean 4.1 ng/ml), whereas amniotic fluids from 50 normal pregnant women contained up to 24 ng GFAP/ml (mean 12.4 ng/ml). GFAP concentrations in CSFs from 32 multiple sclerosis patients were found not to be elevated compared to the control group.     

Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate

Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 x 10(5) cells/mL to 31 x 10(5) cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 x 10(5) cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, q(Mab), continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment. (c) 1993 John Wiley & Sons, Inc.     

PLASMA VASOPRESSIN LEVELS AND WATER CONSERVATION IN FASTING, POSTWEANED NORTHERN ELEPHANT SEAL PUPS (MIROUNGA ANGUSTIROSTRIS)

Northern elephant seal pups ( Mirounga angustirostris ) use several physiological mechanisms to conserve water during their 8- 12-wk postweaning fast. Urine was analyzed from five animals and plasma from four of those animals was sampled serially throughout the postweaning period to quantify changes in urine concentrating ability and electrolyte homeostasis at various stages of the fast (Early = 1-4 wk, Mid = 4-7 wk, Late = 7-10 wk). Plasma osmolarities stayed relatively constant at 301.6 f 13.1 mOsm/kg. Urine osmolarities ranged from 1,053.5 ± 78.6 mOsm/kg (Early), to 1,585.0 ± 136.7 mOsm/kg after eight weeks, dropping to 1,214.3 ± 349.6 mOsm/kg (Late). Urine concentrations for Na⁺ and K⁺ early in the fast were 55.0 ± 14.6 mM and 180.8 ± 48.6 mM, respectively, declining to 8.2 ± 3.3 mM and 91.4 ± 29.0 mM later. Plasma vasopressin concentrations ranged from 34.8 ± 18.2 pg/ml (Early) to 4.8 ± 1.3 pg/ml (Late). The pups appear to conserve their body water by producing a concentrated urine, thus reducing urinary water loss. The significance of the antidiuretic role of vasopressin in the conservation of body water in these pups could not be conclusively determined from the results.     

Chemotherapy of Aspergillus fumigatus keratitis: An experimental study

An experimental Keratitis study of Aspergillus fumigatus was performed in 130 rabbits divided into 12 groups of ten animals each. Three antifungal drugs (myconazole, amphotericin B and pimaricin) were tested using two procedures (topical drops and subconjunctival injections) and two different concentrations (500 and 10 000 times the MIC). In each case, the drugs were applied every 3 h starting 14 h after inoculation. Miconazole was useful at 10 mg/ ml concentration by topical drops and subconjunctival injections, but was less useful at 5 mg/ ml. Amphotericin B was useful at 5 mg/ ml concentration by topical drops and less useful at 2 mg/ ml.No differences were found between the two concentrations by subconjunctival administration. Pimaricin was useful by topical drops at 50 mg/ ml concentration and less useful at 10 mg/ ml as well as by subconjunctival injections.     

Application of capillary electrophoresis to the simultaneous screening and quantitation of benzodiazepines

Capillary electrophoresis (CE) is an attractive approach for the analysis of drugs in body fluids. We made a simultaneous analysis of nitrazepam, diazepam, estazolam, bromazepam, triazolam and flurazepam using CE with on-column detection at 200 nm. We obtained the best electropherograms under a condition of 5 mM phosphate-borate (pH 8.5) containing 50 mM SDS and 15% methanol. We examined the effect of the sample solvent matrix on the electropherograms obtained, indicating that increasing the methanol content in the sample solvent or the injection volume above a certain threshold limit decreased the resolution. We then focused on application of the CE to the analysis of the drugs in spiked serum, being appropriate for an analysis within 25 min. Linearity, the detection limit, accuracy and reproducibility were established using this method. The calibration curve was linear up to 1 mg/l of serum concentration. The lower limit of detection was 5 pg per injection and 0.025 mg/l of the serum concentration for all the compounds except for flurazepam, for which they were 40 pg/injection and 0.2 mg/l. The detection limits obtained allowed toxicological and pharmacological determinations for nitrazepam, diazepam, estazolam and bromazepam, but not for triazolam and flurazepam. Only toxic blood levels for the latter two benzodiazepines could be quantified by this method. We concluded that the CE could at least be applicable to simultaneous screening for toxic levels of benzodiazepines. We suggest that this technique may offer criminal toxicologists a rapid, simple and adaptable approach for the estimation of many other drugs in body fluids.     

The influence of salinity on oxygen consumption and plasma electrolytes in grass carp, Ctenopharyngodon idella Val.

Fingerling grass carp, Ctenopharyngodon idella , 90–160 mm t.l. , were acclimated to experimental salinities of < 1 (fresh water), 91, 195 and 317 mOsm/kg. Oxygen consumption rates of fish declined, as salinity increased, from 0.16 mg O₂/g-h in fresh water to 0.11 mg O₂/g-h at an ambient concentration of 317 mOsm/kg. Plasma electrolytes (Na⁺ and Cl⁻¹) and total plasma ionic concentrations increased slightly following 10 days of exposure to ambient salinities greater than 195 mOsm/kg. At 317 mOsm/kg fish appeared to lose control of plasma electrolyte concentrations. As the osmotic gradient was reduced between the fish and the external medium it might have become advantageous to the maintenance of the osmotic equilibrium to reduce blood circulation to the gills, thus imposing a reduction in metabolic rate.     

Changes in plasma high-density lipoprotein cholesterol concentration after weight reduction in grossly obese subjects.

Changes in serum lipoproteins associated with weight loss were assessed in 13 grossly obese (relative weight 183%) patients who had participated in an outpatient semi-starvation diet consisting of liquid protein and carbohydrate. At the follow-up examination an average of six and a half months after the start of refeeding the mean weight loss was 16.1 +/- 4.5 kg or 15% of initial body weight. Significant increases in high-density lipoprotein (HDL) cholesterol of 0.16 +/- 0.05 mmol/l (6 +/- 2 mg/100 ml) and decreases in triglycerides (0.8 +/- 0.23 mmol/l; 73 +/- 20 mg/100 ml) and fasting blood sugar (0.6 +/- 0.22 mmol/l; 11 +/- 4 mg/100 ml) were observed. Changes in HDL cholesterol correlated significantly with changes in weight (r = 0.667) and percentage change in weight. The intercept of the regression equation relating HDL cholesterol to percentage change in weight was -7.3, indicating that a change in HDL cholesterol greater than zero required a weight loss of at least 7.3% of body weight. Thus, weight loss can significantly increase HDL cholesterol concentrations but a considerable amount of weight must be lost to produce a significant increase in HDL cholesterol concentration.