TIME SEQUENCE OF NUCLEAR PORE FORMATION IN PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES AND IN HELA CELLS DURING THE CELL CYCLE

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/µ² and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from ~2000 in G₁ to ~4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.     

Isoenzyme status and genetic variability of serum esterases in the lesser snow goose,Anser caerulescens caerulescens

A maximum of 22 bands comprising four esterase subgroups—acetylesterase, carboxylesterase, cholinesterase, and acetylcholinesterase—were detected following electrophoresis of lesser snow goose sera on polyacrylamide gels. A minimum of seven structural genes was surmised to be involved in the biosynthesis of these enzymes following physiochemical characterizations. The genetic variability of these loci was calculated to be 1.25% average heterozygosity, while 14.3% of the loci were polymorphic. These estimates of genetic variability were substantially lower than those reported for other vertebrate species. The low degree of genetic variability found in snow goose serum esterases coupled with the extensive protein multiplicity observed may possibly reflect an adaptive strategy based on biochemical plasticity rather than genic heterozygosity for this species. The nature of evolutionary forces acting upon multiple enzyme systems such as esterases is discussed. The concept of conditional neutrality is introduced and defined within this context.This research was carried out under grants from the National Research Council of Canada and the Canadian Wildlife Service to F. Cooke. J. Grossfield was supported by grants from the National Institutes of Health, GM 21630 and FRAP 10576.     

Is Giardia a living fossil?

Despite enormous efforts, the patterns of the rise of eukaryotic life on Earth are not clearly defined. The ability of eukaryotes to produce energy using oxygen and sugars was a key factor in advancing life on Earth towards complex multicellular organisms. However, this was not the only way to produce energy and survive. Mitochondria probably appeared soon after the oxygen increase in the Earth's atmosphere but many microaerophilic protists require little or no oxygen to survive. New ultrastructural, biochemical and molecular phylogeny data about structures and processes involved in the generation of energy by currently known protists have forced the revision of understanding of the "tree of life".     

Isolation, characterization, and identification of Geobacillus thermodenitrificans HRO10, an alpha-amylase and alpha-glucosidase producing thermophile

Thermophilic and amylolytic aerobic bacteria were isolated from soil through a selective enrichment procedure at 60 degrees C with starch as the carbon source. One of the isolates designated as HRO10 produced glucose aside from limit dextrin as the only hydrolysis product from starch and was characterized in detail. The starch-degrading enzymes produced by strain HRO10 were determined to be alpha-amylase and alpha-glucosidase. Whereas the alpha-amylase activity was detected exclusively in the culture supernatant, alpha-glucosidase occurred intracellular, extracellular, or on the surface of the bacteria depending on the growth phase. The optimum temperature and pH required for the growth of strain HRO10 were about 50 degrees C and pH 6.5 to 7.5. The strain used different carbohydrates as the carbon source, but the maximum production of alpha-amylase occurred when 1.0% (w/v) starch or dextrin was used. The use of organic vs. inorganic nitrogen favored the production of alpha-amylase in strain HRO10. The metal ions Li+, Mg2+, and Mn2+ stimulated the production of both enzymes. Identification of strain HRO10 by physiological and molecular methods including sequencing of the 16S rDNA showed that this strain belongs to the species Geobacillus thermodenitrificans. Biochemically, strain HRO10 differs from the type strain DSM 465 only in its ability to hydrolyze starch.     

Determinants of gating polarity of a connexin 32 hemichannel

There is good evidence supporting the view that the transjunctional voltage sensor (V(j)-sensor) of Cx32 and other Group 1 connexins is contained within a segment of the N-terminus that contributes to the formation of the channel pore. We have shown that the addition of negatively charged amino acid residues at several positions within the first 10 amino acid residues reverses the polarity of V(j)-gating and proposed that channel closure is initiated by the inward movement of this region. Here, we report that positive charge substitutions of the 2nd, 5th, and 8th residues maintain the negative polarity of V(j)-gating. These data are consistent with the original gating model. Surprisingly, some channels containing combinations of positive and/or negative charges at the 2nd and 5th positions display bipolar V(j)-gating. The appearance of bipolar gating does not correlate with relative orientation of charges at this position. However, the voltage sensitivity of bipolar channels correlates with the sign of the charge at the 2nd residue, suggesting that charges at this position may have a larger role in determining gating polarity. Taken together with previous findings, the results suggest that the polarity V(j)-gating is not determined by the sign of the charge lying closest to the cytoplasmic entry of the channel, nor is it likely to result from the reorientation of an electrical dipole contained in the N-terminus. We further explore the mechanism of polarity determination by utilizing the one-dimensional Poisson-Nernst-Plank model to determine the voltage profile of simple model channels containing regions of permanent charge within the channel pore. These considerations demonstrate how local variations in the electric field may influence the polarity and sensitivity of V(j)-gating but are unlikely to account for the appearance of bipolar V(j)-gating.     

Characterization and Chromosomal Localization of the Gene for Human Rhodopsin Kinase

G-protein-dependent receptor kinases (GRKs) play a key role in the adaptation of receptors to persistent stimuli. In rod photoreceptors rhodopsin kinase (RK) mediates rapid desensitization of rod photoreceptors to light by catalyzing phosphorylation of the visual pigment rhodopsin. To study the structure and mechanism of GRKs in human photoreceptors, we have isolated and characterized cDNA and genomic clones derived from the human RK locus using a bovine rhodopsin kinase cDNA fragment as a probe. The RK locus, assigned to chromosome 13 band q34, is composed of seven exons that encode a protein 92% identical in amino acid sequence to bovine rhodopsin kinase. The marked difference between the structure of this gene and that of another recently cloned human GRK gene suggests the existence of a wide evolutionary gap between members of the GRK gene family.     

Unbiased transmission of mutant alleles at the human retinoblastoma locus

The preferential transmission of the mutant allele to offspring from fathers who carry a germline mutation in the retinoblastoma gene was examined by analyzing 46 consecutive pedigrees. Among 75 offspring from 29 fathers, the ratio of carriers to noncarriers was 49%. Among the 106 offspring from 55 mothers the ratio was 57%. Neither ratio differs statistically from the expected 50%. When the analysis was limited to only those families with low-penetrance retinoblastoma, we still did not observe a biased transmission of alleles from fathers, although mothers did have an excess of carrier offspring of borderline statistical significance (the P-value was approximately 0.03). While we cannot rule out a biased transmission of alleles from some parents, there appears to be no such bias overall.     

A Highlights from MBoC Selection: Structurally distinct endocytic pathways for B cell receptors in B lymphocytes

B lymphocytes play a critical role in adaptive immunity. On antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to these factors. Here, we studied the mechanism of soluble antigen-induced BCR clustering and internalization in a cultured human B cell line using correlative superresolution fluorescence and platinum replica electron microscopy. First, by visualizing nanoscale BCR clusters, we provide direct evidence that BCR cluster size increases with F(ab’)2 concentration. Next, we show that the physical mechanism of internalization switches in response to BCR cluster size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when cluster size increases beyond the size of a single clathrin-coated pit, B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton.