The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

A thermodynamic study of sperm-egg interaction.

We have studied the binding of spermatozoa to the receptor sites on the vitelline coat (VC) of glycerol-treated eggs (ghost eggs) of the Ascidian, Ciona intestinalis (Protochordate). Glycerol treatment cytolyses the egg without affecting the ability of the VC to bind spermatozoa in a species-specific manner; however, in this system binding is not followed by the acrosome reaction. The ghost eggs are metabolically inert. As a base line for our analysis, we have studied the concentration-dependent heat evolved and oxygen consumption of spermatozoa when diluted in sea water. The process has been analyzed on the basis of equations derived by Liquori and Tripiciano to describe cell growth. Upon binding to the ghost eggs, the spermatozoa produce an explosive heat evolution (excess heat) which is not accompanied by oxygen consumption. The excess heat produced plotted against sperm concentration (at constant egg concentrations) gives an asymmetric bell-shaped curve. This is interpreted as being due to the competitive effect of sperm agglutination at a high sperm concentration. It is concluded that only spermatozoa that attach singly (monomeric spermatozoa) to the egg undergo metabolic activation.     

A conformational analysis of mono and dialkyl ethers of 2,2′-dihydroxy-1,1′-binaphthalene by circular dichroism spectroscopy and cholesteric induction in nematic liquid crystals

The coupling of the analysis of the absorption and circular dichroism (CD) spectra with that of the cholesteric mesophases induced in nematic liquid crystals indicated some interesting conformational features of bridged and nonbridged mono- and dialkylethers of optically active 2,2′-dihydroxy-1,1′-binaphthalene. Bridged derivatives are characterized by relatively small dihedral angles. Simple monoalkyl ethers are characterized by larger dihedral angles but they all assume an s-cis conformation, owing to the existence of intramolecular hydrogen bonds. Nonbridged dialkylethers prefer even larger dihedral angles and, depending on the bulkiness of the alkyl groups, the s-trans conformation can be found. Interestingly, the conformation of dialkylethers is strongly dependent on the structure of the liquid crystal solvent, because the intramolecular hydrogen bond is not possible there. © 1995 Wiley-Liss, Inc.     

Fungicidal activity of Candida albicans-induced murine lymphokine-activated killer cells against C. albicans hyphae in vitro.

Multiple intraperitoneal injections of inactivated Candida albicans cells resulted in the generation of cytotoxic peritoneal cells with phenotypical and functional properties similar to in vitro-generated lymphokine-activated killer (LAK) cells. Using an in vitro [3H]glucose uptake assay, C. albicans-induced LAK-like (CA-LAK) cells exhibited high levels of anti-hyphal activity, the effects being effector to target cell (E:T) ratio- and time-dependent. Maximal levels of anti-C. albicans activity (approximately 60%) were observed after 4 h and at E:T greater than or equal to 300:1. Similar patterns of anti-C. albicans activity were exerted by in vivo-activated natural killer (NK) cells, in vitro interleukin-2- (IL-2) generated LAK cells and polymorphonuclear cells. The anti-hyphal activity of CA-LAK cells was enriched by separation on a Percoll gradient, F2 and F3 fractions retaining most of the activity. Experiments using immunodepressed animals demonstrated that the in vivo lethality of the C. albicans hyphal form is significantly affected by in vitro pre-exposure to CA-LAK cells. While control mice receiving C. albicans alone had a median survival time of 2 d, mice receiving C. albicans pre-exposed to CA-LAK cells (E:T = 300:1) had a median survival time of 15 d. Overall, the susceptibility of the C. albicans hyphal form to CA-LAK cells suggests that C. albicans-induced effectors might play a significant role as a second-line defence mechanism against the C. albicans hyphal form.     

Plant species diversity in Mediterranean old-growth forests: A case study from central Italy

Abstract

To investigate the differences in understorey composition and diversity between old-growth and managed forests, we analyzed an old-growth and a managed beech stand in the same area displaying similar abiotic features. We considered variations in understorey species composition and richness. The sampled understorey species were characterized in terms of functional traits, Ellenberg's indicator values and taxonomic distinctness; next, we calculated four different pairwise plot-to-plot dissimilarity matrices based on species composition, functional traits, Ellenberg's indices and taxonomic distances. We applied a permutational multivariate extension of ANOVA to test whether the forest stands significantly differ in the considered features. Indicator values of all plant species in managed and old-growth stands were evaluated.

The old-growth forest had a higher species richness; permutational analysis of variance showed significant differences between the two stands in plant species composition, functional traits, Ellenberg indices and taxonomic distances. Indicator species analysis highlighted 14 indicator species for the unmanaged stand, while only 3 indicators were found for the managed one.

The results suggest that forest management determines ecological differences that strongly affect plant species composition.

The knowledge of natural stands dynamics could allow development of new approaches and practices in forest management focusing on biodiversity conservation.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies