Expressed Peromyscus maniculatus (Pema) MHC class I genes: evolutionary implications and the identification of a gene encoding a Qa1-like antigen

To gain insight into the evolution of rodent major histocompatibility complex (MHC) class I genes and identify important (conserved) nonclassical class I (class Ib) gene products and residues in these proteins, sixPeromyscus maniculatus MHC (Pema) class I cDNA clones were isolated and sequenced. FivePema class I cDNAs appeared most similar to mouse and rat classical class I (class Ia) genes. One exhibited highest similarity to anH2 class Ib gene,H2-T23 (encoding the Qa1 antigen). Phylogenetic trees constructed withPema, RT1, andH2 class I sequences suggested that the lineages of some rodent class Ib genes (e.g.,T23 andT24) originated prior toMus andPeromyscus speciation [>50 million years (My) ago]. Sequences of four Qa1-like proteins from three species permitted the identification of ten Qa1-specific amino acids. On the basis of molecular modeling, three residues showed the potential to interact with T-cell receptors and three residues (all corresponding to polymorphic positions among H2 class Ia proteins) were predicted to influence antigen binding. The recognition of mouse Qa1 proteins by a subset of T-cells in influenced by a locus,Qdm, which encodes the H2-D leader peptide. One of thePema class I cDNA clones classified asH2-K, D/L-like (class Ia) is predicted to encode an identical peptide, implying that an antigen binding protein (Qa1) and the antigen to which it binds (the product ofQdm) has been conserved for over 50 My. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U12822 (Pm13), U12885 (Pm41), U12886 (Pm52), U12887 (Pm62), U16846 (Pm11), and U16847 (Pm53)     

Pyruvate metabolism in mitochondria from cucumber cotyledons during early seedling development

Mitochondria were isolated from cucumber cotyledons during earlyseedling growth, and their capacity for pyruvate metabolisminvestigated. The rate of pyruvate oxidation was low. Evidenceis presented that suggests that this is due to low activitiesof the pyruvate transporter. Key words: Cotyledon, cucumber, germination, pyruvate oxidation     

H-2T24 and Pema T24: orthologous expressed MHC class Ib genes from mouse and Peromyscus maniculatus

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U03104 and L22338.     

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 -induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.     

Comparison of techniques for measuring plasmid stability in yeasts

Summary Three techniques for measuring plasmid stability in yeasts are described and evaluated. The yeast used was aKluyveromyces lactis strain which was transformed with a plasmid, pCR1, to enable production of heterologous α-amylase. The techniques were based on plate counts on a selective antibiotic-containing medium and a non-selective medium, and on clearing zones on starch-supplemented plates for α-amylase detection. The plate ratio and clearing zones methods gave comparable results while the transfer colony method estimated much lower plasmid stabilities.     

5,7-Diphenyl-3-ureidohexahydroazepin-2-ones as Cholecystokinin-B receptor ligands

A series of 5,7-diphenyl-3-ureidohexahydroazepin-2-one cholecystokinin-B (CCK-B) receptor antagonists was synthesized using Beckmann ring expansion of a suitable 2,4-diphenylcyclohexanone as a key step. SAR studies revealed the importance of the 5-aryl group for high and selective CCK-B receptor affinity, as illustrated in compound (−)-10i (CCK-B IC₅₀ = 6.8 nM).     

The role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain

Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-inactivating proteins (RIPS), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y1 14F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A₁ form of Slt-IA (wild-type-). The same general relationships held: relative to wild type-, Y114F- was attenuated about 7-fold, and Y114S- about 300-fold. Tryptic digestion profiles of the mutant proteins were similar to those of the corresponding wild-type, indicating that the amino acid substitutions had not caused major alterations in conformation. We conclude that Y114 plays a significant role in the activity of Slt-IA, one which is quantitatively similar to that of Y77, and one which is predicated on the presence of both its weakly acidic phenolic hydroxyl and its aromatic ring.     

Expression and secretion of wheat α-amylase in Kluyveromyces lactis

The plasmid pCR1 has been constructed to express a wheat -amylase enzyme in Kluyveromyces lactis strains. The contruct is based on the vector pCXJ-kan1, which has been derived from pDK1, a native plasmid of K. lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Contruct pCR1 produces an -amylase by DNA isolated from a wheat cDNA clone and is controlled by a Saccharomyces cerevisia PGK promoter. Correspondence to: C. Russell     

Causal inference and large-scale expert validation shed light on the drivers of SDM accuracy and variance

Aim

To develop a causal understanding of the drivers of Species distribution model (SDM) performance.

Location

United Kingdom (UK).

Methods

We measured the accuracy and variance of SDMs fitted for 518 species of invertebrate and plant in the UK. Our measure of variance reflects variation among replicate model fits, and taxon experts assessed model accuracy. Using directed acyclic graphs, we developed a causal model depicting plausible effects of explanatory variables (e.g. species' prevalence, sample size) on SDM accuracy and variance and quantified those effects using a multilevel piecewise path model.

Results

According to our model, sample size and niche completeness (proportion of a species' niche covered by sampling) directly affect SDM accuracy and variance. Prevalence and range completeness have indirect effects mediated by sample size. Challenging conventional wisdom, we found that the effect of prevalence on SDM accuracy is positive. This reflects the facts that sample size has a positive effect on accuracy and larger sample sizes are possible for widespread species. It is possible, however, that the omission of an unobserved confounder biased this effect. Previous studies, which reported negative correlations between prevalence and SDM accuracy, conditioned on sample size.

Main conclusions

Our model explicates the causal basis of previously reported correlations between SDM performance and species/data characteristics. It also suggests that niche completeness has similarly large effects on SDM accuracy and variance as sample size. Analysts should consider niche completeness, or proxies thereof, in addition to sample size when deciding whether modelling is worthwhile.     

The efficacy of CGS 20267 in suppressing estrogen biosynthesis in patients with advanced stage breast cancer

The pharmacologic inhibition of aromatase activity has been the focus of clinical trials in patients with advanced stage breast cancer. Recent developments with imidazole compounds that inhibit aromatase activity suggest their clinical use as potent inhibitors of estrogen biosynthesis in postmenopausal breast cancer patients. In this Phase I, open-label, dose-range finding study, we examined the inhibitory potency of CGS 20267 on blood and urine levels of estradiol, estrone and estrone sulfate in 8 patients with metastatic breast cancer. Studies included evaluation of adrenal and thyroid function to look for evidence of general hydroxylase inhibition at dose levels effective for aromatase blockade. Patients were administered CGS 20267 at doses of 0.1 and 0.25 mg, once a day in ascending doses over a 12-week period. Preliminary data reveal that CGS 20267 elicits a striking suppression in plasma estradiol, estrone and estrone sulphate which was observed in some patients as quickly as within 24 h of the first dose. Estrogen suppression of over 90% was achieved within 2 weeks of therapy. No alterations in either baseline or ACTH (cortrosyn) stimulated cortisol and aldosterone levels were observed through the 12 weeks of therapy. In addition, 24 h urine sodium and potassium values were not appreciably altered during therapy. We conclude that CGS 20267 is a potent, specific inhibitor of estrogen biosynthesis in postmenopausal patients with metastatic breast cancer and effectively reduces blood and urine estrogens to undetectable levels.