Calcium-induced modification of the acrosomal matrix in digitonin-permeabilized guinea pig spermatozoa

In this report we describe and partially characterize a preparation of digitonin-permeabilized guinea pig spermatozoa that undergo a rapid and synchronous modification of the acrosomal matrix in response to calcium. Permeabilization of cauda epididymal spermatozoa by digitonin was monitored by using adenylate cyclase activity as an indicator. Spermatozoa (5 x 10(7) cells) treated with 0.005% digitonin for 15 s exhibited maximal adenylate cyclase activity but generally retained their structural morphology, as examined by phase-contrast and transmission electron microscopy. The ratio fo cell number to detergent concentration was the critical factor for determining both the efficiency of permeabilization and the maintenance of structural integrity. When permeabilized spermatozoa were treated with 2 mM CaCl2, the cells underwent a rapid and synchronous modification of the acrosomal matrix (AM). As observed by phase-contrast microscopy, the response to CaCl2 was characterized by events that occurred in the following temporal sequence: disruption of the sperm rouleaux, the loss of refractility by the apical segment of the sperm acrosome, and detachment of the apical segment from the spermatozoa. Transmission electron microscopy indicated that the loss of refractility from the sperm apical segment was coincident with a calcium-induced dispersion of the AM. Analysis of the proteins released during this response, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that a specific subset of sperm proteins was released from the spermatozoa, including a major = staining, 45,000 Mr protein apparently generated from a higher molecular weight precursor during the acrosome reaction.     

The genetic architecture of reproductive isolation in Louisiana irises: pollination syndromes and pollinator preferences

In animal-pollinated plants, pollinator preferences for divergent floral forms can lead to partial reproductive isolation. We describe regions of plant genomes that affect pollinator preferences for two species of Louisiana Irises, Iris brevicaulis and Iris fulva, and their artificial hybrids. Iris brevicaulis and I. fulva possess bee and bird-pollination syndromes, respectively. Hummingbirds preferred I. fulva and under-visited both I. brevicaulis and backcrosses toward this species. Lepidopterans preferred I. fulva and backcrosses toward I. fulva, but also under-visited I. brevicaulis and I. brevicaulis backcrosses. Bumblebees preferred I. brevicaulis and F1 hybrids and rarely visited I. fulva. Although all three pollen vectors preferred one or the other species, these preferences did not prevent visitation to other hybrid/parental classes. Quantitative trait locus (QTL) mapping, in reciprocal BC1 mapping populations, defined the genetic architecture of loci that affected pollinator behavior. We detected six and nine QTLs that affected pollinator visitation rates in the BCIb and BCIf mapping populations, respectively, with as many as three QTLs detected for each trait. Overall, this study reflects the possible role of quantitative genetic factors in determining (1) reproductive isolation, (2) the pattern of pollinator-mediated genetic exchange, and thus (3) hybrid zone evolution.     

The development of exoerythrocytic stages of Plasmodium inui shortti in New World monkeys

Attempts are being made to adapt Old World monkey malarial parasites to New World monkeys for vaccine and molecular studies. Several of these (Plasmodium cynomolgi Berok, Plasmodium fragile, and Plasmodium knowlesi) grow readily but have failed to produce infective gametocytes. Plasmodium gonderi and Plasmodium fieldi develop in the liver after sporozoite inoculation but have failed to establish infection in the erythrocyte. Anopheles dirus mosquitoes infected with Plasmodium inui shortti by feeding on infected macaques transmitted the infection to Saimiri boliviensis monkeys. Infective gametocytes were produced, and sporozoite transmission from Saimiri to Saimiri monkey was obtained. Exoerythrocytic stages have also been observed in the liver tissue of Saimiri monkeys. The availability of the complete transmission cycle provides an additional resource for immunologic and vaccine studies.     

Structural analysis of a set of proteins resulting from a bacterial genomics project

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.     

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.     

Testicular membranes with improved stability of the gonadotropin receptor

A plasma membrane fraction has been prepared from rat testis using an aqueous double-phase polymer system containing dextran, poly(ethylene glycol) 6000 and Zn²⁺. The membrane-associated gonadotropin receptor for lutropin and human choriogonadotropin can be markedly stabilized by a thawing-washing step of frozen membranes which prolongs the apparent half-life of the unoccupied membrane-associated receptors from less than 1 h at 37°C to greater than 5 h. Also, no degradation of ¹²⁵I-labeled human choriogonadotropin was detected following incubation with the membrane fraction. The equilibrium binding was characterized by an apparent association constant of 1.6 · 10¹⁰ M^?1 and a receptor content of 33 fmol/mg protein. Binding kinetic yielded an association rate constant of 1.0 · 10⁸ M^?1, while the dissociation rate constant for human choriogonadotropin was too low to be accurately determined under the conditions used. In contrast, ovine lutropin could be reversibly bound to the membranes leaving the previously occupied receptors available for binding by ¹²⁵I-labeled human choriogonadotropin.