A technique for typing Cryptosporidium isolates.

Antigens extracted from Cryptosporidium oocysts, which had been purified from faeces or chick egg culture, were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels, and blotted onto nitrocellulose membranes. A Cryptosporidium genus-specific monoclonal antibody MAb-C1 bound to multiple bands using several detection techniques, and these corresponded to bands detected using immune rabbit antisera. Using a detection system with fluorescein isothiocyanate (FITC)-labelled MAb-C1 and alkaline phosphatase-labelled anti-FITC, bands were detected between 50 and 300 kDa. Blots were examined directly and by using a laser scanner. The system was shown to be specific for Cryptosporidium spp., giving no staining with a variety of other pathogens, and with negative samples. The oocyst antigen which bound MAb-C1 was stable, and banding patterns were not significantly affected by pretreatment of oocysts with proteinase K, trypsin, formalin, or sodium hypochlorite, methods commonly used during preparation and storage of C. parvum oocysts. However, banding was reduced with potassium dichromate. Of 76 samples containing Cryptosporidium oocysts, 53 showed one or more MAb-C1 staining bands. Cryptosporidium baileyi and C. parvum could be clearly differentiated by their banding patterns, indicating that the system will distinguish between species. Some isolates, including a single isolate of C. muris, produced weak bands which made interpretation difficult. The technique showed differences between isolates of C. parvum, with two different banding types found in human isolates, and other banding types seen in calf and lamb isolates. This method provides a useful way of characterising isolates which may be new species.     

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.     

Pastoral dairy marketing and household wealth interactions and their implications for calves and humans in Ethiopia

Surveys of pastoral households in a semi-nomadic Borana community during 1987–1988 were used to test the hypothesis that poorer families living closest to a market town would be most affected by the enhanced opportunity to sell dairy products, which would intensify competition between people and calves for milk and have negative implications for calf management. These poorer families indeed reported the highest rates of milk offtake per cow, and the milk increment was probably sold to purchase more grain for human consumption at the expense of milk intake for the calf. Consequently, this strategy may increase the susceptibility of malnourished calves to disease, especially those from lower-producing dams. Benefits of improved human energy intake from grain and retention of livestock capital must be weighed against risks of calf death and possible malnutrition of people from milk restriction when assessing dairy marketing trade-offs that are most acute for the poor. Opportunity to sell dairy products at favorable terms of trade helps the poorest people survive, and their risks could be mitigated by policies that facilitate grain marketing in the rangelands and interventions that improve calf feeding management, diversify human diets, and create alternative opportunities for women to generate income. The households postulated to be most at risk were identified from a complex, but logical, interaction among factors of distance to market, household wealth, and the quality of milking cows held. This indicates that targeting such needy groups for development assistance may require a more detailed and interdisciplinary analysis of production systems than is commonly practiced.     

Heterogeneity of apical glycoconjugates in kidney collecting ducts: Further studies using simultaneous detection of lectin binding sites and immunocytochemical detection of key transport enzymes

Summary In the search for a functional role for the polarized glycoconjugates of rat collecting duct epithelial cells, the relation between binding of various lectins and expression of cellular transport enzyme profile of the cells was studied. For this purpose, principal and intercalated cells of rat kidney collecting duct were identified by morphological criteria and by their immunocytochemically determined content of (Na⁺+K⁺)-ATPase and carbonic anhydrase (CA II), respectively. VariousN-acetylgalactosamine-specific lectins such as those fromHelix pomatia andMaclura pomifera revealed heterogeneity among both principal and intercalated cells, whereas -N-acetylgalactosa nine-specific lectin fromDolichos biflorus andVicia villosa bound preferentially to principal cells. Still another lectin fromArachis hypogaea reacted with most collecting duct cells in the cortex and outer medulla, but only with a subpopulation of cells in the inner medulla. Interestingly, some lectins reacted exclusively with the apical aspect of the collecting duct epithelial cells, whereas others revealed both an apical and basolateral distribution of lectin reactive glycoconjugates. The results thus show subtle differences in the glycocalyx structure of principal and intercalated cells and differences in the intracellular polarization of glycoconjugates of these cells. Thus, lectins may be useful tools in the study of the molecular mechanisms which establish and maintain the polarized functions of principal and intercalated cells.     

Intracellular pathway and kinetics of protein secretion in the coagulating gland of the mouse

The coagulating gland of male rodents is part of the prostatic complex. Various mechanisms of secretion have been postulated, in part because organelles commonly involved in the secretory process possess unusual features, such as extreme distension of the rough endoplasmic reticulum. In the present study, the pathway, kinetics, and mode of secretion in the coagulating gland of the mouse were studied by electron microscope autoradiography at intervals between 5 min and 8 h after administration of 3H-threonine. The percentage of grains associated with the rough endoplasmic reticulum was initially high and generally decreased throughout the experiment, while a pronounced rise in the proportion of grains associated with the Golgi apparatus and secretory granules was observed 6 h after injection of precursor. In addition, there was a smaller elevation in the percentage of grains over the Golgi apparatus and secretory granules between 1 and 4 h, and radioactive material first reached the lumen of the gland 4 h after injection of the precursor. Although the general pathway of intracellular transport of secretory protein resembles that in other cells, the results indicate that there are several unusual aspects to the secretory process in the coagulating gland. First, the rate of transport was markedly slower than in most other exocrine gland cells, since the bulk of the labeled protein did not reach the Golgi apparatus and secretory granules until 6 h after administration of precursor. This reflected prolonged retention of secretory products in the endoplasmic reticulum. Second, in addition to the major bolus of labeled material that traversed the cells at about 6 h, a smaller wave of radioactivity appeared to pass through the Golgi apparatus and secretory granules and reach the lumen earlier, within the first few hours after the injection. Finally, the primary mode of secretion in the coagulating gland appears to be merocrine because the secretory granules contained much labeled protein.     

Desorption chemical ionization mass spectrometry of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholines and analogues

Ammonia desorption chemical ionization of ether-linked phospholipids of the type 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (platelet-activating factors) and a series of analogues revealed a systematic fragmentation pattern that is characteristic for these compounds. The predominant ions included the protonated molecular ion and a series of fragments derived from the molecular ion having the following nominal mass losses: MH-14, MH-42, MH-59, and MH-183. Deuterated ammonia was used to elucidate the nature of several fragments. In addition, desorption chemical ionization was used to quantitate 1-O-hexadecyl-2-O-acetyl-sn-glycero-3-phosphocholine at the nanogram/sample level.     

Mechanism of interferon action. Expression of vesicular stomatitis virus G gene in transfected COS cells is inhibited by interferon at the level of protein synthesis

The effect of interferon on the expression of the vesicular stomatitis virus glycoprotein G gene was examined in simian COS cells transfected with the expression vector pSVGL containing the G gene under the control of the SV40 late promoter. When COS cells were treated with interferon 24 h after transfection, the synthesis of vesicular stomatitis virus G protein was inhibited by about 80% as compared to that in untreated controls. By contrast, under the same conditions, neither the plasmid copy number nor the G gene mRNA levels were detectably affected by interferon treatment. Likewise, the synthesis of simian virus 40 large T-antigen was not inhibited by interferon treatment of transfected COS cells even though the synthesis of vesicular stomatitis virus G protein was markedly inhibited. The residual G protein synthesized in transfected, interferon-treated COS cells appeared to be normally glycosylated.     

Crystallization of insecticyanin from the hemolymph of the tobacco hornworm Manduca sexta L. in a form suitable for a high resolution structure determination

Insecticyanin, a blue biliprotein from the tobacco hornworm Manduca sexta, has been crystallized in a form suitable for a high resolution x-ray analysis. The crystals grow by vapor diffusion against solutions of polyethylene glycol 8000 at pH 5.5. They belong to the space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 115.0 A; c = 71.1 A. Insecticyanin is believed to be a tetramer in solution; there are two subunits per asymmetric unit. The crystals diffract to at least 2.2 A resolution and appear reasonably resistant to radiation damage.     

Release of Endogenous Amino Acids from Striatal Neurons in Primary Culture

Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.