Horizontal polyacrylamide gradient gel electrophoresis for the simultaneous phenotyping of transferrin, post-transferrin, albumin and post-albumin in the blood plasma of cattle

A simple method of horizontal polyacrylamide gel electrophoresis was described for the simultaneous phenotyping of transferrin, post-transferrin, albumin and post-albumin in the blood plasma of cattle. A step gradient gel of 8, 4, 12 and 14% acrylamide concentration was used. The method enabled the detection of a new protein polymorphism in the post-transferrin region. Two alleles were observed. The transferrin phenotypes involving D1 and D2 alleles were clearly separated. The resolution of the post-albumin fractions was also better than described by earlier methods.     

Polymorphic post-albumin of cattle and horse plasma identified as vitamin D binding protein (Gc protein)

Cattle and horse plasma samples of known post-albumin types were radiolabelled with ¹⁴C-vitamin D₃. These samples were then analysed by polyacrylamide gel electrophoresis, followed by autoradiography. The patterns observed were identical to those of post-albumin variants. The polymorphic post-albumin protein of cattle and horse was thus identified as the vitamin D binding protein and homologous to the Gc protein of human plasma.     

Two-dimensional gel electrophoresis of sheep plasma proteins: Genetic polymorphism of an ai-protease inhibitor and a post-transferrin

Two-dimensional electrophoretic analysis of sheep plasma proteins was performed by a first-dimension separation in agarose gel (pH 5.0) followed by a second-dimension one in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many α- and β-globulins. Two groups of α₁-globulins, designated Pi-1 and Pi-2, were found to be protease inhibitors. These two inhibitors differed from each other in protease inhibitory spectra. Genetic polymorphism was observed for the Pi-2 protein and another unidentified protein, tentatively designated as post-transferrin (Ptf). Family data supported the hypothesis that Pi-2 and Ptf types were controlled by codominant, autosomal alleles. Three Pi-2 alleles and two Ptf alleles were observed in one population of the Gotland breed of sheep. The analysis of data from 50 informative matings showed no evidence of genetic linkage between the Pi-2, Ptf and transferrin (Tf) loci in the population of sheep studied.     

Coat colour inheritance in Soay, Orkney and Shetland sheep

Analysis of the numerical proportions of Soay, Orkney and Shetland sheep of different colours together with test matings, produced results compatible with the hypothesis that these breeds have a multiple allelic series at locus A , white ( A ₁) being dominant to grey ( A ₂) and both being dominant to the gene for self-colour ( A ₅). The alleles at the A locus are epistatic to the alleles for pigment production at locus B , black ( B ₁) being dominant to brown ( B ₂).     

Genetic polymorphism of horse serum protein 3 (SP3)

Summary. Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles ( D,F,I,S ). Evidence was provided that the F allele can be further divided into two alleles ( F ₁ and F ₂); the mobilities of F₁ and F₂ variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.     

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing*

Summary. Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe₀-transferrin, Fe₁-transferrin and Fe₂-transferrin region. At the level of Fe₀-transferrin and Fe₁-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.     

Genetic polymorphism of the vitamin D binding protein (Gc) in the musk shrew (Suncus murinus)

In the musk shrew ( Suncus murinus ), the electrophoretic bands in the post-albumin region were identified as vitamin D binding protein (Gc) by the [³HI vitamin D₃ binding method. Three Gc phenotypes were distinguished from each other: a single faster band (Gc-A), a single slower band (Gc-B) and the double bands (Gc-AB). Results of mating experiments indicated that the Gc-A and Gc-B are controlled by two codominant alleles, Cc ^a and Gc ^b at an autosomal locus ( Cc ), respectively. It was noticed that, in the Gc-AB phenotypes, the Gc-B band was constantly more intense than the Gc-A band in the protein staining. The same tendency was also observed btween the homozygous Gc-A and Gc-B bands, and further, radioactivity of the Gc-B bound with [³H] vitamin D₃ was about twofold higher than that of the Gc-A. These results suggest that the Gc^b yields its protein product twofold more than the Gc ^a. No cross-reaction between the shrew proteins and a rabbit anti-human Gc protein was observed.     

Allele frequencies of the major milk proteins in the Finnish Ayrshire and detection of a new K-casein variant

A total of 20990 Finnish Ayrshire cows were phenotyped for the major milk proteins by isoelectric focusing in polyacrylamide gels. The predominant alleles in the Finnish Ayrshire were α_S1-casein B (0.999), α_S2-casein A (0.991), β-casein A₁ (0.509) and α₂ (0.490), α₂₂-casein A (0.612) and β-lactoglobulin B (0.716). The K-casein E allele (0.307) was also rather common in the Finnish Ayrshire. A new K-casein variant (K-casein F) was demonstrated in two Finnish Ayrshire cows, a dam and a daughter.     

Effects of river flow on larval growth and survival of Japanese seaperch Lateolabrax japonicus (Pisces) in the Chikugo River estuary, upper Ariake Bay

The abundance and growth history of larval and juvenile Japanese seaperch Lateolabrax japonicus were investigated in the Chikugo River estuary, upper Ariake Bay, from 1990 to 2000. Growth during the larval period (up to 15 mm standard length, L _S, the size at recruitment into the estuary) was backcalculated using sagittal otolith microstructures by the biological intercept method. Growth rates in length declined at body sizes >14 mm L _S. High freshwater discharge through the Chikugo River was associated with high temperatures of the upper Ariake Bay where the larvae spend their planktonic life. Mean larval stage duration (days) from hatch to 15 mm ( D ₁₅) varied between 48·8 and 76·2 days and was inversely correlated with the estimated mean temperature history [mean daily temperature (° C) experienced by the larvae during the period from hatch to 15 mm, T ₁₅]. Mean abundance (number m⁻²) of larvae and juveniles was highest in years when T ₁₅, D ₁₅ and freshwater discharge were at intermediate levels. Although the abundance was not correlated with either of these variables, an exponential relationship between abundance and D ₁₅ was found when data collected during the highest river discharge years (1990, 1991 and 1998) were excluded. The increase in freshwater discharge through the Chikugo River probably had the potential to enhance or diminish Japanese seaperch recruitment in two ways: 1) it could increase recruitment probability by increasing temperature and larval growth and 2) high river flow also had the potential to decrease the probability of immigration into the river by increasing larval seaward dispersion, predation due to decreased turbidity and starvation due to decreased zooplankton prey abundance in the estuary.     

Seasonal changes in condition indices in adult mature and non-maturing burbot, Lota lota (L.), in the north-eastern Bothnian Bay, northern Finland

The population structure and seasonal changes in condition factors of the burbot in a shallow coastal region of the north-eastern Bothnian Bay are described. The significance of the so-called rest years is examined by comparing condition indices in immature or sterile and mature burbot. The somatic condition index ( K ₂), liver index ( K ₁), intestine index ( K ₁) and gonad index ( K_g ) are determined monthly in terms of organ weight in relation to body length. Approximately 30% of the whole catch of 1052 burbot were non-maturing but were 40 cm or more in length. K ₂, K ₁ and K ₁ were lowest in the autumn, when the first sign of gonad recrudescence was observed in mature burbot. The non-maturing burbot were never in poorer condition than mature ones. As mature and non-maturing burbot dissipated their energy stores during the warmest period of the summer, it is concluded that burbot spending a rest year do not accumulate and store energy reserves over the summer for the next year, and that such rest years, if they exist, do not occur for nutritional reasons.     

Behavioural and physiological adaptations to a burrowing lifestyle in the snake blenny, Lumpenus lampretaeformis, and the red band-fish, Cepola rubescens

Observations have been made on the mode of burrow construction in the snake blenny, Lumpenus lampretaeformis , under laboratory conditions. It appears that head probing and lateral oscillations of the body are principally responsible for the excavation of the burrow which is completed within 24 h. The burrow structure has been analysed in detail, showing a mean depth of 7.2 cm with a maximum observed length of 73 cm, with most systems between 20 and 35 cm in length. Initially linear burrows with two openings are usually provided with a small side tunnel, giving the system a characteristic Y-shape.
Burrow irrigation was investigated for the first time in L. lampretaeformis. The mean duration of burrow irrigation, by flexions of the tail of the fish, was 21 s with over 13 min h⁻¹ spent in irrigating the burrow. The mean water displacement per irrigation period was 3.1 ml. The PO ₂ and PCO ₂ were measured in both surface water and within the burrow system of L. lampretaeformis. Surface water values for PO ₂ were high (> 150 Torr) and PCO ₂ low (<0.4 Torr). Hypoxic and hypercapnic conditions were measured in the burrow system itself, with PO ₂ values ranging between 57 and 129 Torr and PCO ₂ rising to > 1.3 Torr in some burrows.
A comparative study of Cepola rubescens burrows indicated similar surface water PO ₂ and PCO ₂ values as in L. lampretaeformis. Burrow water PO ₂ values ranged between 60 and 94 Torr, with PCO ₂ values as high as 1.5 Torr being recorded. These results are discussed in relation to the adaptation of both species to a burrowing lifestyle.     

Stomatal response to humidity: implications for transpiration

Abstract. Transpiration rates from apple leaves are analysed in terms of the ratio of latent heat flux (λ E ) to leaf net radiation ( Q ₁) and the climatological resistance ( r_i ). Increases in stomatal resistance with increasing leaf to air vapour pressure gradient ( D ), described by an empirical model, are incorporated in the analysis. This humidity effect causes the proportion of energy dissipated as latent heat to fall as Q ₁ increases, so that leaf transpiration rates in high energy environments are likely to be similar to those in lower energy environments. Boundary layer resistance ( r _a) exerts an increasingly important effect on transpiration rates as Q ₁ increases. At constant Q ₁ stomatal closure in response to increasing D results in very small changes in leaf temperature ( T ₁) across a wide range of ambient vapour pressure deficits (δ e ); r _a is then the major factor determining T ₁. The implications of these results are discussed.     

Purification and N-terminal amino acid sequence of Enterocin CRL 35, a 'pediocin-like' bacteriocin produced by Enterococcus faecium CRL 35

M.E. FARÍAS, R.N. FARÍAS, A.P. DE RUIZ HOLGADO AND F. SESMA. 1996. Enterocin CRL 35, a bacteriocin produced by Enterococcus faecium CRL 35 that inhibits food-borne pathogens, was purified by precipitation with (NH₄)₂SO₄, gel filtration, ion exchange and reverse phase chromatography. The partial N-terminal amino acid sequence indicated a strong homology with other 'pediocin-like bacteriocins' previously described.     

Sensitivity of some common grain fungi to irradiation on grain and in phosphate-buffered saline

The sensitivity of nine cereal fungi to irradiation on grain and in phosphate-buffered saline (PBS) was investigated. Species of Fusarium and Alternaria were more resistant to irradiation (higher D ₁₀ value) than Aspergillus spp. and Penicillium spp. Generally D ₁₀ values determined on grain were lower than the corresponding values measured in PBS. The plating medium did not significantly affect the D ₁₀ values.     

NADP+-dependent glutamate dehydrogenase from the facultative methylotroph Hyphomicrobium X

Abstract NADP⁺-dependent glutamate dehydrogenase (GDH; EC 1.4.1.4) was purified using acetone precipitation, heat, DEAE-cellulose and dye-ligand Ramazol Red column chromatography. The M _r of the native enzyme was estimated to be 380 000 (± 10 000) by polyacrylamide gel electrophoresis. The same technique in the presence of sodium dodecyl sulphate (SDS) gave one subunit band with an M _r of 63 400 (±4000). Thus the enzyme has a hexameric structure. The enzyme has a pH optimum of 8.5 and has K _m apparent values of 1.6 mM, 0.015 mM and 10.2 mM for α-ketoglutarate, N NADPH and L -glutamate, respectively. Michaelis-Menten kinetics were not observed when the ammonium concentration was increased. A progressive increase in the ammonium concentration resulted in a progressively increasing K _m value. The enzyme was highly specific for all substrates and markedly insensitive to inhibitors.     

Application of the 'Inoculated Pack Test' method for the determination of the heat resistance of Clostridium sporogenes PA3679 spores in acidified mushrooms

The effect of pH in the range 5.2–6.7 on the thermal destruction of Clostridium sporogenes PA3679 spores suspended in mushrooms in brine acidified with citric acid was examined by the 'inoculated pack test' method. The results indicated that increasing acidity is accompanied by decreasing decimal reduction times at 121.1°C: D _121.1 at pH 6.0 and 5.2 was, respectively, 64% and 17.5% of that at pH 6.7, the pH of natural mushrooms ( D _121.1= 2.22 min). A linear model ( r = 0.988, α= 0.05) was developed where the D _121.1 value was a function of the pH over the range studied. The inoculated pack test seems to be the only method to evaluate the actual microbial heat resistance, whether of spore or of vegetative forms, in order to estimate within reasonably close limits a suitable process time required to eliminate health hazards and to prevent spoilage losses in a given food product.     

Two tertiary branched tetraamines, N4-aminopropylnorspermidine and N4-aminopropylsperimidine, are the major polyamines in thermophilic eubacteria, Thermoleophilum

Abstract Two novel polyamines were found as major polyamines of Thermoleophilum album and Thermoleophilum minutum , which are Gram-negative eubacteria obligate for thermophily and n -alkane substrates. They were identified as tertiary branched tetraamines, N⁴-aminopropylnorspermidine ( tris (3-aminopropyl)amine) [NH₂(CH₂)₃N-((CH₂)₃NH₂)(CH₂)₃NH₂ or N(CH₂CH₂CH₂NH₂)₃] and N⁴-aminopropylspermidine [NH₂(CH₂)₃N((CH₂)₃NH₂)(CH₂)₄NH₂] by high-performance liquid chromatography and gas chromatography-mass spectrometry.     

Two new sheep transferrin variants and the effect of neuraminidase

Transferrin phenotypes were determined in six breeds of sheep by starch gel electrophoresis. Two new variants, Tf H_Czech and Tf K_Czech, were found and some evidence of their genetic control was obtained. Tf H_Czeeh was detected only in Sumava sheep; it has an intermediate mobility between Tf A and Tf B. Tf K_Czech was found only in Tsigais; it was localized between Tf B and Tf C. The frequencies of corresponding alleles were very low.
Individual transferrin variants (I, A, H_Czech, B, K_czech, C, D, E, and P) were treated with neuraminidase. Electrophoretic mobility of the strong band was decreased by two steps in each case. It suggests that in the strong Tf band two sialic acid residues are accessible to the enzyme.     

A METHOD FOR MEASURING THE NARCOTIC ACTION OF CHEMICALS USING THE GRAIN WEEVIL, CALANDRA GRANARIA

A method of measuring narcotic potency is described. Grain weevils ( Calandra granaria ) are exposed to the narcotic vapour in 2l. round-bottomed flasks illuminated from the bottom, and narcotic potency measured as number of grain weevils so affected as to be unable to climb the glass walls. Some common anaesthetics have been examined as an illustration of this method. Results are fitted by the probit plane
Y= A + b ₁ log α+ b ₂ log t ,
where Y = a function of the number of insects affected (probit); α= the thermodynamic concentration of the narcotic vapour; t = time of exposure of insects to the narcotic vapour. The values of the coefficients b ₁ and b₂ are discussed in relation to the biological action of the narcotic vapours.