Investigation of the Booroola (FecB) and Inverdale (FecX(I)) mutations in 21 prolific breeds and strains of sheep sampled in 13 countries.

Twenty-one of the world's prolific sheep breeds and strains were tested for the presence of the FecB mutation of BMPR1B and the FecX(I) mutation of BMP15. The breeds studied were Romanov (2 strains), Finn (2 strains), East Friesian, Teeswater, Blueface Leicester, Hu, Han, D'Man, Chios, Mountain Sheep (three breeds), German Whiteheaded Mutton, Lleyn, Loa, Galician, Barbados Blackbelly (pure and crossbred) and St. Croix. The FecB mutation was found in two breeds, Hu and Han from China, but not in any of the other breeds. The 12 Hu sheep sampled were all homozygous carriers of FecB (FecB(B)/FecB(B)) whereas the sample of 12 Han sheep included all three genotypes (FecB(B)/FecB(B), FecB(B)/FecB+, FecB+/FecB+) at frequencies of 0.33, 0.58 and 0.08, respectively. There was no evidence of FecX(I) in any of the breeds sampled.

Cloning and characterization of a G protein alpha-subunit-encoding gene from the basidiomycete, Coprinus congregatus.

Complementary DNA (cDNA) clones encoding two G protein alpha-subunit proteins (CGP alpha 1 and CGP alpha 2) were isolated from a Coprinus congregatus (Cc) hyphal tip cell (HTC) library using PCR-generated biotinylated G protein probes. Sequence analysis of the Cc cgp alpha 1 gene indicates that the gene contains an open reading frame (ORF) that translates into a putative 353-amino-acid (aa) product. The predicted CGP alpha 1 protein exhibits similarity to all known G protein alpha-subunits (it has all of the consensus regions for a GTP-binding protein), especially the mammalian retinal G protein, transducin. The CGP alpha 1 aa sequence is 50% identical overall to the transducin subfamily, cgp alpha 1 shares the same aa size grouping as transducin alpha-subunits and, unlike many other G proteins, both CGP alpha 1 and transducin seem to possess a cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive site. Preliminary reverse transcription PCR (RT-PCR) analysis of cgp alpha 1 and cgp alpha 2 mRNA expression revealed that, unlike cgp alpha 2 which seems to be constitutively expressed, cgp alpha 1 is expressed only in HTC that are competent in responding to light. Thus, the cgp alpha 1 product, CGP alpha 1, is a likely candidate for regulating the blue light-induced signal transduction photomorphogenesis system found in Cc.

Differential expression of milk protein genes during lactation in the common brushtail possum (Trichosurus vulpecula).

In the common brushtail possum (Trichosurus vulpecula) lactation lasts for 200 days and consists of two distinct phases. Milk composition changes dramatically between phase 2 and 3, which correspond to early and late lactation respectively (phase 1 corresponds to pregnancy). RNA expression patterns have been established for eight major milk protein genes throughout lactation in possum mammary glands. The levels of mRNA expressed from two genes, encoding the early and late lactation proteins, were differentially regulated during lactation, with peak RNA levels occurring in phase 2 and 3 of lactation respectively. Expression of these two RNA transcripts did not overlap, and neither gene was expressed at significant levels between days 116 to 125, suggesting that the transition from phase 2 to phase 3 of lactation occurs at this time. The level of lysozyme, alpha-lactalbumin and trichosurin mRNA increased in phase 3 of lactation, whereas the levels of beta-lactoglobulin, alpha-casein and beta-casein mRNA remained constant throughout lactation. In the non-suckled gland, expression of milk protein genes was greatly reduced by day 6 of lactation. In conclusion, the early and late lactation protein genes are good markers for phase 2 and 3 of lactation, with the transition between these phases occurring around day 120 of lactation in the possum.

A rapid method for cosmid cloning.

We present a method for genomic library construction using cosmid vectors. With a combination of backfilling with Klenow enzyme and a cosmid vector with two cos sites, a DNA bank in excess of 500,000 clones can be made from 10 micrograms of genomic DNA. The method is more rapid than conventional protocols because size fractionation of target DNA is not necessary. A further advantage is that libraries can be made from relatively small amounts of genomic DNA.

Single-trunk anomalous origin of both coronary arteries from the pulmonary artery. Diagnosis and surgical management.

The cases of two infants with heart failure and myocardial infarction because of single-trunk anomalous origin of both coronary arteries from the pulmonary artery are reported. Electrocardiography and thallium 201 imaging indicated preoperative myocardial infarction. The diagnosis was confirmed by cardiac catheterization and angiography in each case. To our knowledge these are the first reports of this diagnosis being made during life prior to attempts at surgical correction. Both patients underwent cardiac operations and the operative techniques used are described. Corrective operations for this abnormality have not been attempted previously. At autopsy radiopaque contrast material injected into the aorta confirmed flow from the aorta to the coronary arteries.

The costs of coronary artery surgery to the community.

The possible benefits obtained both for the community and patients following coronary artery bypass grafting (CABG) have been examined in 4001 patients (mean age 54 years, 14.8% female) who underwent CABG from 1971 to 1982 in the Cardiothoracic Surgical Unit of the Royal Adelaide Hospital. Assessment followed a fixed response format questionnaire sent to all patients with a total follow-up of 98.8%. The hospital mortality was 1.4% for the 10 year period with a fall from 4% in 1973 to 0.9% in 1981. Shifts in employment were assessed in survivors. At follow up, 548 patients who were previously employed had ceased full-time or part-time employment. This decrease in employment was attributed mainly to age. Analysis of retirement curves for the general male population and male patients showed a similar pattern but downward translation of 4% for the pre-operative group. A similar downward shift occurred following surgery. For female patients, performance of home duties improved from 159 patients on full home duties pre-operatively to 292 post-operatively. Visits to the doctor dropped in 50% of surviving patients. CABG has a low mortality. The community benefits by helping to maintain productivity and decreased cost for ongoing medical care.

A 4-year experience with the Björk-Shiley Monostrut valve.

We reviewed 314 consecutive patients in whom a Björk-Shiley Monostrut aortic valve prosthesis was inserted by our cardiothoracic surgical unit from June 1982 through June 1986. The group comprised 220 males (70%) and 94 females (30%), with a mean age of 60 years (range, 5 to 83 years). Two hundred ninety-six patients (94%) received an aortic prosthesis alone, and the other 18 (6%) received both an aortic Monostrut valve and a Starr-Edwards mitral valve prosthesis. In each case, preoperative data were collected prospectively, and a questionnaire was sent at the time of follow-up. The median follow-up period was 23 months (range, 0 to 46 months); 6 (2%) of the patients were lost to follow-up. In the remaining 308 cases, there were 20 deaths (6.5%), 8 of which occurred within approximately 1 month after operation. There were no known instances of valve failure. One death occurred in the group that underwent double-valve replacement. Valve endocarditis accounted for 2 late deaths. With respect to preoperative dyspnea, 59 (19%) of the patients were in New York Heart Association functional Class I, 74 (24%) were in Class II, 138 (45%) were in Class III, and 37 (12%) were in Class IV. Postoperatively, there were 247 patients (80%) in Class I, 43 (14%) in Class II, 15 (5%) in Class III, and 3 (1%) in Class IV (p < 0.0001). All of the patients received postoperative anticoagulant therapy; 3.4% per year were subsequently readmitted for bleeding problems. Thromboembolic events necessitating hospitalization occurred in 3.0% of the patients per year. On the basis of these results, our early experience with the Björk-Shiley Monostrut valve in the aortic position has been satisfactory, with an acceptable rate of complications and no documented valve failures.

The genetic basis for the meiotic disorder of Coprinus congregatus.

A "pale-mushroom" phenotype in the fungus Coprinus congregatus has previously been reported to be caused by a cytoplasmic, infectious agent that inhibits the formation of synaptonemal complexes. After a period of 5 years of successful extraction of infectious material from carrier homokaryons, infectivity has now been lost and even cytoplasmic contact through somatic fusions no longer transmits the pale phenotype. In attempts to obtain new carrier homokaryons, a genetic analysis has been done. This study demonstrates that the pale phenotype in this species is primarily determined by a nuclear gene, symbolized pal (-). The pale phenotype appears predictably when the dikaryon is homoallelic for the pal (-) allele. Homoallelic wild type (pal (+)) and heteroallelic dikaryons display the dark phenotype. The former infectious nature of the pale phenotype and the current anomalous behavior of one homokaryon is discussed in the context that the pale phenotype may involve both a nuclear gene and a cytoplasmic element under some conditions.

The effect of proteases on plasmodium formation in Didymium iridis.

An improved assay for quantitatively measuring the number of plasmodia formed with time is presented. Using this assay we have investigated the effects of three proteases, subtilisin PBN', subtilisin carlsberg and alpha-chymotrypsin. We have shown that 1) plasmodium formation is sensitive to protease treatment only during the first 2 h after mixing amoebae of compatible mating type but not after, 2) amoebae are protease sensitive when treated 1 h prior to mixing, 3) the two clones used have different sensitivities to protease treatment and 4) these effects are due to enzymatic activity and have little effect on viability. The meaning of these results in relation to recent evidence for a diffusible inducer of plasmodium formation is discussed.

Signal transduction in Coprinus congregatus: evidence for the involvement of G proteins in blue light photomorphogenesis.

This paper reports the presence of several G proteins and light-sensitive GTP-binding proteins in the fungus Coprinus congregatus, a filamentous eukaryote. (Mono)ADP-ribosylation experiments with crude membranes in the presence of the (poly)ADP-ribosyltransferase inhibitor, 3-amino-benzamide, resulted in the detection of a cholera toxin substrate of 52 kDa and two pertussis toxin substrates, 33 and 39 kDa. Two-dimensional polyacrylamide gel analysis of GTP-binding proteins exposed in vivo to [35S]-labeled guanosine 5'-[gamma-thio]-triphosphate in the presence or absence of light demonstrated light enhanced analog binding. These results support the concept of the involvement of G proteins in phototransduction in C. congregatus.

Angiotensin II stimulates glucose transport activity in cultured vascular smooth muscle cells.

The glucose transport system in cultured rat vascular smooth muscle cells has been examined by measuring the uptake of 2-deoxyglucose. Angiotensin II (Ang II) stimulated 2-deoxyglucose uptake in cells made quiescent by removing serum from the culture medium in a dose- and time-dependent manner that was shown to be receptor-mediated. Epidermal growth factor (EGF), fetal calf serum, thrombin, and arginine vasopressin also stimulated glucose transport. Cycloheximide did not affect the immediate-early (30 min) activation by either Ang II or EGF, but abolished any further increase. This suggested that, whereas the initial activation of glucose transport was independent of protein synthesis, the sustained increase required the synthesis of new glucose transporters. This was supported by 4-fold and 2-fold accumulations of GLUT-1 mRNA 4 h after exposure to Ang II and EGF, respectively. The induction of GLUT-1 mRNA was preceded by rapid and transient expression of c-fos and c-jun protooncogenes. In nuclear run-on assays, nuclei from Ang II-treated cells showed increased synthesis of GLUT-1 mRNA at 30 min and 1 h after hormone treatment. In contrast, in cells exposed to actinomycin D, pretreatment with Ang II had no effect on the turnover rates of GLUT-1 mRNa. These results are consistent with Ang II acting to stimulate the rate of transcription of the GLUT-1 gene leading to increased production of GLUT-1 protein and glucose transport.

Glucose deprivation and acute cycloheximide treatment stimulate system L amino acid transport in cultured vascular smooth muscle cells.

The effect of glucose deprivation on the uptake of leucine has been examined in cultured vascular smooth muscle cells isolated from rat aortae. Equimolar substitution of sucrose or fructose for glucose in the culture medium enhanced the uptake of leucine in a time- and concentration-dependent manner. The effect was first detectable after 12 h and reached the maximum, 2-fold, after 48 h with an apparent half-maximal effect at 1 mM glucose and could be reversed after 48 h of glucose refeeding. The enhanced leucine uptake was completely inhibited by 2-amino-2-norbornane-carboxylic acid, a specific substrate for System L, but not by alpha-(methylamino)isobutyric acid or lysine. Kinetic analyses indicated that this stimulation was mediated via a homogenous system with a 1.7-fold increase in the Vmax without any change in the Km (0.15 mM). Prolonged treatments with cycloheximide (10 micrograms/ml) or actinomycin D (10 micrograms/ml) blocked this glucose deprivation effect and its reversal. However, cycloheximide also very rapidly stimulated leucine uptake, reaching the maximum, 2.5-fold over the basal at 1 h. This effect occurred at concentrations that matched its inhibition on protein synthesis (half-maximal at 0.1 micrograms/ml) and could be reproduced with puromycin as well as actinomycin D. The stimulatory effect of cycloheximide was also accompanied by an increase in the Vmax but not in the Km, being sensitive to 2-amino-2-norbornane-carboxylic acid inhibition only, and appeared to occur in an additive manner to that of glucose deprivation. Although the uptake of leucine was stimulated by glucose deprivation and brief exposure to cycloheximide, these treatments had no effect on the efflux of the substrate. These results are all consistent with the System L amino acids transport activity in cultured rat vascular smooth muscle cells being under the control of at least two non-hormonal regulatory mechanisms, one that is likely to involve a labile repressor molecule and the other involving de novo protein synthesis as a result of chronic glucose deprivation.

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells.

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a Na+ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y+ transporter.

Changes in isozyme patterns between monokaryons and dikaryons of a bipolar Coprinus.

Proteins and isozymes of several different classes of enzymes in partially purified protein extracts of monokaryons, dikaryons, and monokaryon mixtures of a bipolar Coprinus sp. were separated on polyacrylamide gels by slab electrophoresis. Differences in protein and isozyme spectra were correlated with the operation of the incompatibility factors and with the results of Wang and Raper on Schizophyllum. It was concluded that the shift from monokaryon to dikaryon mediated a major change in the nature, quantity, or distribution of the proteins of this Coprinus sp.

A highly infectious 'mycoplasma' that inhibits meiosis in the fungus Coprinus.

We have discovered a cytoplasmically inherited infectious agent that inhibits meiosis in a species of Coprinus, a basidiomycetous fungus. From infectivity, filtration, centrifugation and ultrastructural studies we believe the agent to be a mycoplasma. The agent is highly infectious to several strains of the host species and is capable of spreading rapidly through infected hosts. No pathological effect has been seen on any aspect of growth or differentiation of the fungus except for the inability of infected strains or undergo meiosis. The failure of meiosis results in mushrooms that do not produce the normal black spores and are therefore pale in colour. The paleness represents a simple assay for the presence and activity of the infectious agent. Infected hosts do not display any ultrastructural abnormalities in the vegetative stages, only in the cells in which meiosis should occur. In the meiotic cells, at the time when normal cells are undergoing synapsis and synaptinemal complexes are forming, the vacuoles of the infected cells become occupied with vesicular, membrane-bound bodies resembling in shape and form mycoplasmas. Extracts from infected clones may be filtered through 0.2-mum filters and retain full infectivity. The infectious material may be pelleted from such extracts at only 10 000g. Migration experiments, as well as the filtration studies, rule out involvement directly of nuclei. The high rate of infection and spread of the mycoplasma through the host, combined with the anatomical simplicity of the host, make this an ideal system in which to study the basis of infection. The singularity of the pathological effect make this host-parasite association useful in studying both the underlying mechanisms of mycoplasma pathogenicity and to investigate the regulation of meiosis. This is only the second report of mycoplasmas in fungi.

The secreted aspartate proteinase of Candida albicans: physiology of secretion and virulence of a proteinase-deficient mutant.

It was established that Candida albicans grew rapidly in a simple medium containing yeast extract (0.2%, w/v) plus glucose (2%, w/v). These cultures were in or near to a state of nitrogen limitation and the concentration of secreted aspartate proteinase increased rapidly (within 3-4 h) on addition of BSA. Synthesis and secretion were apparently controlled both positively (induction by albumin or, more probably, the peptides produced from it) and negatively (repression by NH4Cl). A small intracellular pool of the enzyme was detected during production of the enzyme and this pool decreased with the cessation of synthesis and secretion. A stable mutant, IR24, was isolated which secreted less than 0.3% of the amount of the proteinase exported by the parent strain ATCC 10261. The LD50 values for mutant IR24 and the parent strain administered intravenously to mice were greater than 1.0 x 10(9) and 1.6 x 10(6) c.f.u. kg-1 respectively.