Immunological recognition of the prolactin receptor: identification of a single binding unit of molecular weight approximately 42,000

Different polyclonal and monoclonal antibodies against the rabbit mammary prolactin (PRL) receptor were previously obtained that totally inhibited PRL binding in the rabbit mammary gland. Only polyclonal antibodies were shown to immunoprecipitate preformed PRL--receptor complexes in solubilized mammary membranes suggesting that they also recognized domains outside of the PRL binding site of the receptor. When partially purified PRL receptor preparations from both rabbit and pig mammary tissues were iodinated, immunoprecipitated and subsequently analyzed by SDS--PAGE, a single component of molecular weight approximately 42,000 was specifically recognized by all the anti-PRL receptor antibodies. This unit was the only component immunoprecipitated by the monoclonal antibody M 110. Its identification was not impaired by using reducing or non-reducing conditions. Moreover, a further purification of the [125I]-labeled receptor preparations from both species by a second PRL affinity chromatography selected a single binding unit of the same molecular weight. In contrast, polyclonal antibodies immunoprecipitated additional components apart from the 42,000 unit, especially one unit of molecular weight 70,000-80,000 in both species. We conclude that rabbit and pig mammary PRL receptors exhibit striking immunological similarities. Both contain a single binding unit of molecular weight approximately 42,000 that is not linked to other units via disulfide bridges. This binding unit could be associated with a larger component of MW 70,000-80,000 in the holo receptor.     

Determination of the serological sex-specific (Sxs) antigen (“H-Y antigen”) in birds and mammals using high-titer antisera and a sensitive urease ELISA

Summary A rapid, sensitive, and reproducible enzyme-linked immunosorbent assay (ELISA), for the detection of the serological sex-specific (Sxs) antigen (formerly termed H-Y antigen; see Introduction), is described. This assay uses bovine lestes extract as the solid phase antigen, and high-titer anti-Sxs antisera and a urease-conjugated anti rat-IgG as the first and second antibody respectively. The urea containing substrate causes a pH shift in a positive reaction, which in turn is visualized by the use of bromocresol purple as a pH indicator. The method, and some representative applications of it, are described in detail.     

Fish cleaning behaviour of shrimp

Cleaning behaviour of five species of shrimp from three families was studied at three different geographic locations in an effort to gain a quantitative understanding of cleaning behaviour, and to compare a broad cross-section of cleaner shrimp species. Two shrimp from the genus Periclimenes , two from the genus Lysmata , and one from the genus Stenopus were used and 27 hours of recorded laboratory observations were made for each of the five shrimp species.
All shrimp species were inactive most of the observed time, and most spent less than 2% of the observed time cleaning fish hosts. Also, the shrimp spent more time cleaning the ventral rather than the dorsal surface of the fish because they were reluctant to board the fish. However, evenness in cleaning does not appear to be an indicator of overall excellence in cleaning because the two best cleaners (based on number and duration of cleaning bouts) were among the least even in their cleaning.
The fish cleaning behaviour of the shrimp appeared to be strictly stereotypic in form, but the stimulus-response and the total amount of cleaning differed greatly among the five species. A Cleaning Efficiency Index (CEI) was created in an attempt to incorporate significant aspects of the cleaning behaviour. According to this CEI, Lysmata grabhami was by far the most efficient (best) cleaner, CEI = 55–51, compared to the others; Stenopus hispidus , 33–78; Periclimenes pedersoni , 6–29; Periclimenes yucatunicus , 5–60; and Lysmata californien , 2–12.
The cleaners most widely distributed geographically have the highest CEI scores, while the most localized cleaners have the lowest CEIs. This relationship may allow the CEI score to be useful in determining a cleaner shrimp's potential geographical distribution, and may also serve as an indicator for the degree of phylogenetic relationship to other cleaner shrimps.     

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically

Summary The areA ^r -18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5 and 3 moieties. Surprisingly, we have selected rare intracistronic revertants of areA ^r -18. From crosses heterozygous for areA ^r -18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5 moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3 portion of areA and that the 5 portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and in frame initiation codons to the 3 portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.     

Interactions between developmental processes,growth, and food selection in the larvae and juveniles of Rutilus rutilus (L.) (Cyprinidae)

Summary The aim of this study was to assess the effects of developmental events, occurring in fish during the first weeks after hatching, on the quantity and quality of the ingested food and on growth. The investigation was carried out with the larvae and juveniles of Rutilus rutilus, the single cyprinid species occurring in an oligotrophic subalpine lake in Tirol, Austria. Comparison between availability of prey in the water and gut contents suggests that the selection of food by the young fish is strongly influenced by developmental processes. For example, the prevalence of indigestible phytoplankton in the gut of young larvae can be taken as a sign of the not yet fully developed sensory and locomotory capacities of the young fish (El-Fiky et al. 1987). Furthermore, quantitative and qualitative changes in the gut contents correlate strongly with changes in the form and relative length of the gut, but reflect only weakly the availability of prey in the water. In the Seefelder See population of R. rutilus the switch from a phytoplankton to a cladoceran dominated diet is accompanied by an increase in relative growth rate by nearly one order of magnitude (Wieser et al. 1988).     

Sequence similarities between chicken intestinal 110-kDa ATPase and myosin I-like enzymes

We report the partial amino acid sequence of chicken intestinal microvillar 110-kDa protein that, as a complex with calmodulin, has previously been shown to exhibit myosin-like ATPase and actin-binding activities. The sequence shows a high degree of similarity to the sequence of a novel vertebrate myosin I-like heavy chain encoded by a cDNA isolated from bovine intestine. This confirms that the bovine and chicken proteins are the first examples of Acanthamoeba myosin I-like proteins from higher eukaryotes. Comparison of available structural and functional data leads us to postulate that the myosin I family of proteins result from the fusion of a conserved myosin headlike motor domain, with variable COOH-terminal domains responsible for binding to specific intracellular structures.     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.     

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.