Biological and serological procedures to detect three nepoviruses in fruit trees

Cherry leaf roll virus (CLRV), Myrobalan latent ringspot virus (MLRSV) and Strawberry latent ringspot virus (SLRSV) were transferred by budding to woody trees, hybrid Ishtara, peach cv. GF 305 and cv. Lesiberian. Three buffers with antioxidants and stabilisers: 0.01M phosphate with 1% caffeine; 0.007M phosphate-0.01M veronal with 0.01M cysteine hydrochloride and 0.007 EDTA; 0.015M phosphate with 1% nicotine and 0.066M phosphate buffer without additives were compared for their efficiency in mechanical transmission from woody sources to herbaceous hosts (Chenopodium quinoa and C. amaranticolor). 0.007M phosphate-0.01M veronal buffer with 0.01M cysteine hydrochloride, and 0.007 EDTA and 0.015M phosphate buffer with 1% nicotine were found to be the best buffers for the three nepoviruses. Both biological transmission to herbaceous assay hosts and detection by ELISA in the investigated tree are necessary to reliably detect the three nepoviruses. Biological detection is reliable from April to June, and in September and October. ELISA detection is also more difficult in July and August. The suitability of C. quinoa and C. amaranticolor to maintain CLRV, MLRSV and SLRSV was compared. C. amaranticolor plants were found to be more suitable for CLRV and SLRSV, infected plants grow over 6 months after mechanical inoculation by the nepoviruses. C. quinoa plants proved to be most suitable for maintenance of MLRSV, while C. amaranticolor is a symptomless host of MLRSV. Reinoculation with the nepoviruses is recommended in intervals of 4 to 6 months.

Reduction of Campylobacter jejuni in a Simulated Chicken Digestive Tract by Lactobacilli Cultures

Studies were conducted to investigate the impact of a selected lactobacilli mixed culture on Campylobacter jejuni in simulated chicken digestive tract models. Veronal buffer solutions corresponding to the pH of successive segments of the chicken digestive tract were prepared. The lactobacilli mixtures were prepared by mixing four fresh lactobacilli cultures, including Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus crispatus, and Lactobacillus brevis. The C. jejuni and lactobacilli mixture were mixed with sterile poultry feed, and the previously prepared veronal buffer solutions were then added separately. The mixture was incubated at 41.1°C for various lengths of time with periodic agitation. The feed passage time for five segments of the digestive tract were adopted: crop (pH 4.5), 30 min; proventriculus (pH 4.4), 15 min; gizzard (pH 2.6), 90 min; small intestine (pH 6.2), 90 min; and large intestine (pH 6.3), 15 min. The Campylobacter and lactobacilli were enumerated. An antagonistic effect on C. jejuni by the tested lactobacilli spp. was found in individual sections and the complete simulated digestive tract models. In the simulated complete chicken digestion system, no C. jejuni were found during the final incubation period when a lactobacilli mixture was present. The results of this in vitro study indicate the potential value of future in vivo studies.

Some qualitative differences of hCG in serum from early and late pregnancies and trophoblastic diseases

Twenty-four sera were obtained from women in the first and third trimester of pregnancy, women with gestational choriocarcinoma, and men with testicular hCG-producing tumours. The hCG-activity was measured by an in vivo bioassay (B) based upon the increase in mouse uterine weight. The sera were also analysed by three immunoassays (I), one for hCG, one for α-hCG subunits, and one for β-subunits. All sera were also subjected to electrophoresis in 0.17% agarose suspension in 0.075 mol/l sodium veronal buffer at pH 8.6, and median charge, expressed as median mobility, was estimated for hCG. The metabolic clearance rate (MCR) of hCG in serum in early and late pregnancy and from one patient with choriocarcinoma was measured in mice. The B/I ratio was lower and the forms of hCG were less negatively charged in late pregnancy compared with early pregnancy. The lower B/I ratio might be due to the higher MCR of hCG in the test animal in the in vivo bioassay. The B/I ratio in choriocarcinoma was higher than in early pregnancy. However, the median charge of hCG was almost identical in these two groups. The most negatively charged forms of hCG were found in the men with testicular tumours. The four groups: early pregnancy, late pregnancy, gestational choriocarcinoma, and testicular tumours could be differentiated by measuring the median charge and the B/I ratio of hCG in serum. The production, later in pregnancy, of forms of hCG which disappear faster from the circulation might partially explain the lower hCG concentration in serum at this stage of gestation.

EFFECT OF pH DURING PREPARATION OF EUGLOBULIN PRECIPITATES ON t-PA OR UK ASSAY IN PLASMA

A more sensitive method for the commonly used euglobulin method is proposed for examining plasminogen activators (PAs) in blood, since little attention has been paid to the conditions of production of euglobulin precipitates, especially the pH during their preparation. In the present study, the effect of pH on measurement of the activity of tissue-type plasminogen activator (t-PA) or urokinase (UK) in plasma was investigated and compared by enzymography, bio-immunoassay (BIA) and the fibrin plate method. The antigenicity of PAs was observed as follows. Polyclonal anti-t-PA or UK rabbit IgG previously purified with aprotinin-Sepharose to remove protease activity in the IgG fraction (final concentration, 0.375 mg/ml of 0.01% Triton X-100) was reacted with euglobulin precipitate at 4°C for 1 hr. After incubation, enzymography was preformed, and the residual fibrinolytic activity was measured qualitatively. Euglobulin precipitates were prepared by dilution (1:20) of fresh venous occlusion plasma obtained from healthy volunteers (n = 5) with ice cold, distilled water, and adjusted to pH 4.5 - 7.5 with 0.1% (v/v) acetic acid. The euglobulin precipitates were collected by centrifuge at 4°C, and dissolved in Veronal buffer (0.1 M NaCl, 0.05 M barbital Na, pH 7.75) to the original volume. The PA activities in these solutions were examined by the above three methods. The fibrinolytic activities between pH 5.0 and 6.8 were found to be almost the same (no statistical difference) by the fibrin plate method. However, a stronger t-PA activity as determined from the molecular weight as well as antigenicity, was detected at pH 6.7 by enzymography and BIA. On the other hand, a stronger UK activity as determined from the molecular weight and antigenicity was recognized at pH 5.6 by enzymography. The present results suggest, therefore, that it is necessary to consider the effect of pH during the preparation of euglobulin precipitates for the measurement of t-PA or UK assay in plasma. The suitable pH value for t-PA and UK assays in plasma is 6.7 and 5.6, respectively.

The regulation of metabolic clearance rate of human FSH in mice by variation of the molecular structure of the hormone

The relationship between charge on human FSH (hFSH) molecules and their metabolic clearance rate (MCR) was investigated. The median charge of the hFSH molecules was expressed as their median mobility at electrophoresis in 0.075 m sodium veronal buffer, pH 8.6. MCR was estimated after single iv injection in mice of unfractionated and fractionated extracts of human pituitaries. There was a highly significant (P < 0.001) correlation between charge and MCR both for forms of FSH present within the individual pituitary and for FSH in different pituitaries. After the iv injection there was a gradual change to a more negative median charge of hFSH in plasma. This was explained by the more rapid clearance of the less negatively charged forms of hFSH and thus selective survival of different forms in the circulation. This is a most likely explanation for the differences in median charge between FSH in pituitaries and in sera of men and women of corresponding age. The results suggest that MCR of human FSH is controlled by a gonadal-pituitary feed-back mechanism which involves changes in the structure of the FSH produced by the pituitary. It is suggested that charge is one factor involved in the regulation of the survival of FSH in the circulation and that this is of physiological importance for the control of the ovarian function during the menstrual cycle.

Median charge and charge heterogeneity of human pituitary FSH, LH and TSH

The charge and charge heterogeneity of FSH, LH and TSH in 63 extracts of individual human pituitaries were investigated by use of zone electrophoresis in 0.17% agarose suspension in veronal buffer at pH 8.6 followed by hormone analyses by radioimmunoassay. The migration velocities are similar to those in free solution and directly proportional to differences in charge in the buffer. The technique gave highly reproducible results and the recovery of hormone activity after electrophoresis was about 100%. Methods for estimation of median charge, expressed as median migration rate, and of degree of charge heterogeneity are described. Each pituitary contained a sequence of different forms, estimated at 20 or more, of each of the three hormones, with minor differences in charge. The degree of charge heterogeneity within the pituitary was similar for FSH, LH and TSH. The distribution curves of radioimmunological activity in relation to migration rate were close to normality for the three glycoprotein hormones. This was in contrast to that found for Prl and GH. The merits of the electrophoretic technique are discussed.

A New Factor Control Plasma For Establishing Calibration Curves For Factor-Assay In Extrinsic And Intrinsic System

We present a lyophilised human plasma which is optimal for calibration curves and factor control in extrinsic as well as in intrinsic system. Comparative assays were carried out between factor control plasma and a fresh plasma pool which was frozen at -70°C from 15 normal young men. The fresh plasma pool and the factor control plasmas for the calibration curves were diluted with Owren’s veronal buffer pH 7,35 in a geometric sequence. All deficiency plasmas (American Dade, Division of American Hospital Supply Corporation) were used according to the instructions of the manufacturer. All assays were carried out by two independant investigators.For the extrinsic system Thromboplastin C and FS (American Dade) were used. With this thromboplastin the callibration curves for the fresh plasma pool and the factor control plasma showed an identical course when plotted semilogarithmically. The calibration curves for the factors II, V, VII, and X showed a good correlation to normal plasma pool.For the intrinsic system the PTT-reagent Actin (American Dade) was used. The measuring times for the calibration curves of the factor control plasma are slightly longer than of the fresh plasma pool.When factors determined in factor control plasmas are read from the fresh plasma calibration curves, the range of normal values is from 90 to 105% for the factors in extrinsic system and from 80 to 105% in intrinsic system. According to these assessments, the factor control plasma seems to be appropriate for establishing calibration curves for factor assays as well as for calibration of thromboplastin time. This is a contribution to further standardization of coagulation assays.

Dispersion of Stabilized Plasma Clots Incubated in Normal Plasma or Serum by 5 M Urea

Clots obtained from normal native platelet-poor plasma are dispersed by addition of 5 M urea within 120 hours. Clots from plasma diluted > : 4 (Ann. NY. Acad. Sei. 202, 341–343, 1972) and clots from undiluted plasma, thoroughly washed and incubated in veronal buffer pH 7.4, are not dispersed by urea, but they are rendered susceptible to the dispersing action of urea by prior incubation in normal undiluted plasma or serum. The same phenomenon was observed by incubating the otherwise unsoluble clots in normal plasma or serum fractions precipitated at 33% saturation with (NH4)2SO4. The plasma fraction is more active than the serum one. This fraction was tested by casein-14C putrescine Factor XIII assay: it does not interfere with the incorporation of putrescine into casein, although it conditions the urea- dispersing action on stable clots.