In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Persistent effects of phorbol 12-myristate 13-acetate: Possible implication of vesicle traffic

Relative to their normal counterparts, transformed epithelial cells have a distinctive and quantifiable three-dimensional shape. Biophysical and mathematical methods are used to distinguish these extremes in cells from two lines, cultured from rat liver and tracheal epithelium, respectively. Cells adopted a more transformed-looking configuration transiently when exposed to phorbol 12-myristate 13-acetate (PMA) (Plummer and Heckman, [1990] Exp. Cell Res., 188:66–74). The purpose of the present work was to dissect the physiological processes involved in the shape change. Ruffling activity, known to be PMA-stimulated in other cells, was investigated. Although the ruffles appeared less robust than normal, PMA stimulated ruffling activity over a 5 h period. The number of sites where ruffling was initiated declined by 5 h, however, and suppression was seen by 10 h. Cells from both lines adopted the transformed shape configuration when exposed for 2 h to monensin. When the subset of shape features changed by this treatment was compared with those originally changed during transformation, it was found that monensin-treated cells mimicked the features of transformed cells. Its effect on ruffling was, however, unlike PMA's. Thus, the phenotype was unlikely to arise from ruffling itself but might be a process driven by ruffling. Chloroquine also stimulated cells to assume characteristics of transformed cells. Since both it and monensin could interfere with endosomes and with the processing of endocytosed contents, this was a likely site of action. Experiments were done to determine whether PMA also affected the processing of extracellular fluids. When the accumulation of horseradish peroxidase (HRP) was measured, the rate was found to be higher in PMA-treated cells from 5 min, the earliest time assayed, onward. The results suggest that the transformed type of cell in these cell lines showed a constitutive dilation and/or reorganization of some portion of the endosomal pathway. © 1996 Wiley-Liss, Inc.     

Analysis of a bone assemblage made by chimpanzees at Gombe National Park, Tanzania

Chimpanzee hunting provides information on prey characteristics and constraints acting on a large-bodied primate lacking a hunting technology, and has important implications for modeling hunting by fossil hominids. Analysis of the remains of five red colobus monkeys captured and consumed by Gombe chimpanzees in a single hunting bout provides one of the first opportunities to investigate the characteristics of prey bones surviving chimpanzee consumption. Four of the five individuals (an older infant, two juveniles and one subadult) were preserved in the bone assemblage; a neonate was entirely consumed. Cranial and mandibular fragments had the highest survivorships, followed by the scapulae and long bones. Post-cranial axial elements had the lowest survivorships. A high percentage (80%) of the long bones and ribs surviving consumption were damaged, most commonly through crenulation and step fracturing of bone ends. One of two partially reconstructed crania preserves a canine puncture through its left parietal. Proposed characteristics of faunal assemblages formed through chimpanzee-like hunting include small modal prey size, limited taxonomic diversity, a high proportion of immature individuals and a high frequency of skull bones. These characteristics would not uniquely identify hunting by fossil primates in the geological record, necessitating a contextual approach to diagnose hunting by hominids not forming an archeological record.Hominid utilization of vertebrate tissue is first unambiguously documented at 2.5 m.y.a. Rather than representing a strict "scavenging phase" in the evolution of hominid-prey interactions, Oldowan hominid carnivory may represent the overlay of large mammal scavenging on a tradition of small mammal hunting having a low archeological visibility.     

Persistence of high density lipoprotein particles in obese mice lacking apolipoprotein A-I

Obese mice without leptin (ob/ob) or the leptin receptor (db/db) have increased plasma HDL levels and accumulate a unique lipoprotein referred to as LDL/HDL1. To determine the role of apolipoprotein A-I (apoA-I) in the formation and accumulation of LDL/HDL1, both ob/ob and db/db mice were crossed onto an apoA-I-deficient (apoA-I(-/-)) background. Even though the obese apoA-I(-/-) mice had an expected dramatic decrease in HDL levels, the LDL/HDL1 particle persisted. The cholesterol in this lipoprotein range was associated with both alpha- and beta-migrating particles, confirming the presence of small LDLs and large HDLs. Moreover, in the obese apoA-I(-/-) mice, LDL particles were smaller and HDLs were more negatively charged and enriched in apoE compared with controls. This LDL/HDL1 particle was rapidly remodeled to the size of normal HDL after injection into C57BL/6 mice, but it was not catabolized in obese apoA-I(-/-) mice even though plasma hepatic lipase (HL) activity was increased significantly. The finding of decreased hepatic scavenger receptor class B type I (SR-BI) protein levels may explain the persistence of LDL/HDL1 in obese apoA-I(-/-) mice. Our studies suggest that the maturation and removal of large HDLs depends on the integrity of a functional axis of apoA-I, HL, and SR-BI. Moreover, the presence of large HDLs without apoA-I provides evidence for an apoA-I-independent pathway of cholesterol efflux, possibly sustained by apoE.     

Sequence and peptide-binding motif for a variant of HLA-A*0214 (A*02142) in an HIV-1-resistant individual from the Nairobi Sex Worker cohort

As part of the ongoing study of natural HIV-1 resistance in the women of the Nairobi Sex Workers' study, we have examined a resistance-associated HLA class I allele at the molecular level. Typing by polymerase chain reaction using sequence-specific primers determined that this molecule is closely related to HLA-A*0214, one of a family of HLA-A2 supertype alleles which correlate with HIV-1 resistance in this population. Direct nucleotide sequencing shows that this molecule differs from A*0214, having a silent nucleotide substitution. We therefore propose to designate it HLA-A*02142. We have determined the peptide-binding motif of HLA-A*0214/02142 by peptide elution and bulk Edman degradative sequencing. The resulting motif, X-[Q,V]-X-X-X-K-X-X-[V,L], includes lysine as an anchor at position 6. The data complement available information on the peptide-binding characteristics of this molecule, and will be of use in identifying antigenic peptides from HIV-1 and other pathogens.     

Male vocal imitation produces call convergence during pair bonding in budgerigars, Melopsittacus undulatus

The budgerigar, a small species of parrot, can learn new vocalizations throughout life and is therefore widely used as a model system for studying various aspects of vocal learning. It is not known, however, why parrots imitate sounds. To test the hypothesis that vocal imitation in budgerigars is related to pair bonding, we recorded approximately 100 contact calls from each of nine male and nine female adult budgerigars that were unfamiliar with one another and then placed them into pairs. We sampled their contact call repertoire weekly and conducted twice-weekly behavioural observation sessions. We compared contact calls by sonagram cross-correlation and classified them by means of a hierarchical clustering algorithm. This analysis showed that all pairs developed a shared call within an average of 2.1 weeks. Further analysis revealed that eight of the nine male budgerigars imitated the contact calls of their assigned mates, while none of the females imitated the calls of their males. We conclude that contact call imitation in adult budgerigars probably contributes to pair bond formation and maintenance. Prior studies on budgerigars were limited by the lack of a behavioural paradigm to elicit vocal imitation reliably. Our study remedies this and thereby serves as a foundation for future studies on vocal learning in adult animals. Copyright 2000 The Association for the Study of Animal Behaviour.     

High pH reversed‐phase chromatography as a superior fractionation scheme compared to off‐gel isoelectric focusing for complex proteome analysis

MS/MS is the technology of choice for analyzing complex protein mixtures. However, due to the intrinsic complexity and dynamic range present in higher eukaryotic proteomes, prefractionation is an important step to maximize the number of proteins identified. Off‐gel IEF (OG‐IEF) and high pH RP (Hp‐RP) column chromatography have both been successfully utilized as a first‐dimension peptide separation technique in shotgun proteomic experiments. Here, a direct comparison of the two methodologies was performed on ex vivo peripheral blood mononuclear cell lysate. In 12‐fraction replicate analysis, Hp‐RP resulted in more peptides and proteins identified than OG‐IEF fractionation. Distributions of peptide pIs and hydropathy did not reveal any appreciable bias in either technique. Resolution, defined here as the ability to limit a specific peptide to one particular fraction, was significantly better for Hp‐RP. This leads to a more uniform distribution of total and unique peptides for Hp‐RP across all fractions collected. These results suggest that fractionation by Hp‐RP over OG‐IEF is the better choice for typical complex proteome analysis.