An Acetic Acid Dissociation, Air-Drying Technique for Insect Chromosomes, With Aceto-Lactic Orcein Staining

The method differs from mammalian techniques for somatic chromosomes in that it uses very small amounts of material. Drosophila melanogaster and an ant, Dorymyrmex sp., are used as examples. Pretreatment with 0.05% Colcemid in insect Ringer solution is applied to mature Drosophila larvae for 5 hr, by feeding, but Dorymyrmex prepupae require puncture and a 15 hr exposure of the puncture to the solution. Organs are removed under 1% sodium citrate, tansferred to fresh citrate for 10-20 min, than fixed in acetic-methanol, 1:3, for 30 min. Transfer to a drop of 60% acetic acid on a clean warmed slide dissociates the cells, which are spread by adding a small drop of fixative and tilting the slide in all directions. After immersion in acetic ethanol, 1:3, for 4 hr, rinsing in the stain solvent and draining the slides then have 2-3 drops of aceto-lactic orcein placed on each, coverslips added, and warmed (at about 50 C) for about 12 hr or until staining is sufficient. They can then either be treated as semipermanent or made permanent by allowing the coverslips to slide off in acetic-ethanol, dehydrating, and mounting in Euparal, or a synthetic resin.     

A Differential Staining Technique for Simultaneous Visualization of Mitotic Spindle and Chromosomes in Mammalian Cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanobacetic acid solution containing 4 mM MgCl₂ and 1.5 mM CaCl₂ at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

Movement of Naegleria fowleri Stimulated by Mammalian Cells In Vitro1

ABSTRACT. Naegleria fowleri amebae, but not those of N. australiensis, N. gruberi, or N. lovaniensis, demonstrated enhanced motility when placed in proximity to mammalian cells. Amebae of nonpathogenic species of Naegleria, however, were more motile in cell culture medium than the amebae of N. fowleri. The locomotory response of highly pathogenic mouse-passaged N. fowleri amebae to nerve cells was greater than axenically cultured amebae. The enhanced mobility elicited by whole nerve cells or disrupted nerve cells was not directed migration but chemokinetic. Naegleria fowleri responded to disrupted neuroblastoma cells more vigorously than to disrupted African green monkey kidney (Vero) cells.     

An Easy-To-Handle Microfluidic Device Suitable for Immunohistochemical Procedures in Mammalian Cells Grown Under Flow Conditions

Microfluidics, the technology that manipulates small amount of fluids in microscale complex devices, has undergone a remarkable development during the last decade, by targeting a significant range of applications, including biological tests and single-cell analysis, and by displaying many advantages such as reduced reagent consumption, decreased costs and faster analysis. Furthermore, the introduction of microfluidic tools has revolutionized the study of vascular functions, because the controlled three-dimensional environment and the continuous perfusion provided by the microdevice allow simulating the physiological characteristics of the circulatory system. Researchers interested in the study of vascular physiology, however, are often hampered by the difficulty in handling reduced number of cells after growth in these devices. This work shows how to apply different protocols commonly used in biology, such as the immunofluorescence technique, to cells grown in reversibly-bound microfluidic devices, obtaining results comparable to those retrieved under static conditions in multiwells. In this way, we are able to combine the advantages of microfluidic, i.e., application of continuous flow and shear stress, with classical protocols for the study of endothelial cells.     

Double Fluorescent Staining for the Separate Demonstration of Chromosomes and Microtubules in Mitotic Cells In Vitro

A simple fluorescent method for double staining of mitotic cells using a rhodamine B indirect immunofluorescent method for tubulin and the DNA-specific fluorescent dye Hoechst 33258 for nuclei and chromosomes is described. This procedure enables one through the use of appropriate excitation filters to view at will either chromosomes and nuclei or tubulin within the same cell.     

Simple Methods for Mammalian Chromosomes

The ordinary Feulgen-squash technic, after acetic-alcohol fixation, provides a simple and reliable method of preparing many mammalian tissues for chromosome counts. Tissues best suited to the technic were the seminiferous tubules. Small pieces of tissue about 3-6 mm. long and 1-2 mm. wide were immersed in a freshly made fixative (composed of 3 parts absolute ethyl alcohol and 1 part glacial acetic acid) immediately after removal by biopsy or from a freshly killed animal. After fixation for 3-12 hours the tissue was stained by the standard Feulgen procedure after hydrolysis for 12 minutes in normal HCl at 60 °C A 1-2 mm. piece was then teased apart on a slide in 45% acetic acid with a pair of mounted needles, and the teased tissue was squashed between the slide and the cover slip. During squashing the pressure was applied by hand and was regulated so as to avoid any excessive scattering of the chromosomes. The preparations were made permanent by dehydrating and mounting in Euparal.     

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells¹. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of ''cellular microbiology''². Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories^3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 2787⁵, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787ΔaidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before⁶.Download video file.^{(34M, mov)}     

簇毛麦染色体的改良C—分带

运用改良的C-分带技术,使簇毛麦(Haynaldia villosa Schur)V染色体组的7对染色体全部显示出特异性的丰富带纹,包括与N-带相似的近着丝粒带以及N-带所不具有的末端带和亚端带。这些带纹使V组7对染色体之间以及簇毛麦染色体与小麦染色体之间能明确地相区分。     

Gas Exchange of In Vitro and Ex Vitro Grown Grapevine Plants

Net photosynthetic rate (P _N) and dark respiration rate (R _D) were measured in Vitis vinifera L. cvs. Dimiat 4/24 (23^rd subculture), Dimiat 4/38 (22^nd subculture), and Italian Riesling 3/47 (22^nd subculture) on days 3, 2, and 1 (1^st series) before transfer from the in vitro culture and on days 14, 15, 16 (2^nd series) and 28, 29, 30 (3^rd series) after the transfer. P _N of in vitro and ex vitro plants was strongly affected by irradiance. P _N and R _D of in vitro plantlets were lower and transpiration rate (E) was higher compared to those of ex vitro plantlets. P _N, R _D, and E changed in the course of acclimation.     

Merkel Cells as Putative Regulatory Cells in Skin Disorders: An In Vitro Study

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca²⁺-independent, as inhibition of Ca²⁺ channels or culture in the absence of Ca²⁺ failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated.     

Non-Exponential Growth By Mammalian Cells In Culture

The concept of exponential growth by mammalian cells in culture is based upon the apparent linearity of semilogarithmic data plots. This method of graphical analysis is known to be an unreliable test of the exponential hypothesis. We have re-examined the question of growth exponentiality using the more sensitive method of Smith plots, in which specific growth rate is plotted against either time or density on transformed graphical coordinates which linearize the mathematical expression of the growth hypothesis being tested. With exponential growth, data points fall on a horizontal straight line when specific growth rate is plotted against time or density. Using both our own and literature data, we have performed Smith plot analyses on the growth of 125 different mammalian and avian cell lines. of these, only eleven exhibited an exponential phase. the remaining cell lines all had non-exponential growth patterns. the most common of these consisted of an initial period of growth acceleration followed by a later phase of deceleratory growth. A smaller number of lines exhibited deceleratory kinetics at all times after plating. We conclude that mammalian cell growth in culture is predominantly non-exponential, and that the apparent exponentiality of semilogarithmic data plots is usually a methodological artifact.     

Differential Incorporation of Cholesterol by Sindbis Virus Grown in Mammalian or Insect Cells

Cholesterol has been shown to be essential for the fusion of alphaviruses with artificial membranes (liposomes). Cholesterol has also been implicated as playing an essential and critical role in the processes of entry and egress of alphaviruses in living cells. Paradoxically, insects, the alternate host for alphaviruses, are cholesterol auxotrophs and contain very low levels of this sterol. To further evaluate the role of cholesterol in the life cycle of alphaviruses, the cholesterol levels of the alphavirus Sindbis produced from three different mosquito (Aedes albopictus) cell lines; one other insect cell line, Sf21 from Spodoptera frugiperda; and BHK (mammalian) cells were measured. Sindbis virus was grown in insect cells under normal culture conditions and in cells depleted of cholesterol by growth in serum delipidated by using Cab-O-sil, medium treated with methyl-β-cyclodextrin, or serum-free medium. The levels of cholesterol incorporated into the membranes of the cells and into the virus produced from these cells were determined. Virus produced from these treated and untreated cells was compared to virus grown in BHK cells under standard conditions. The ability of insect cells to produce Sindbis virus after delipidation was found to be highly cell specific and not dependent on the level of cholesterol in the cell membrane. A very low level of cholesterol was required for the generation of wild-type levels of infectious Sindbis virus from delipidated cells. The data show that one role of the virus membrane is structural, providing the stability required for infectivity that may not be provided by the delipidated membranes in some cells. These data show that the amount of cholesterol in the host cell membrane in and of itself has no effect on the process of virus assembly or on the ability of virus to infect cells. Rather, these data suggest that the cholesterol dependence reported for infectivity and assembly of Sindbis virus is a reflection of differences in the insect cell lines used and the methods of delipidation.Sindbis virus, the prototypic Alphavirus, assembles highly symmetrical particles with an associated membrane of host cell origin. The infectious particle is composed of two nested icosahedral shells of T=4 geometry with an intervening membrane bilayer (41). The three structural proteins which comprise the particle, E1, E2, and capsid, are found in a 1:1:1 stoichiometric ratio. The outer shell, composed of glycoproteins E1 and E2, and the nucleocapsid are associated with the outer protein shell through specific interactions of the E2 endodomain with the capsid protein (28-30, 53). Both E1 and E2 are anchored into the membrane bilayer by transmembrane domains (44). During maturation of the virus, the glycoproteins E1 and E2 are processed and oligomerize into trimers of heterodimers and are delivered to the cell surface by the cellular exocytic pathway (6, 39). In mammalian cells, the glycoproteins are trafficked to the plasma membrane to unite with preformed nucleocapsids (5, 12). The maturational pathway used by this virus in insect cells is also via the exocytic pathway; however, in these cells, the virus particles assemble within cytoplasmic vesicles which release virus directly into the extracellular medium (4, 38). It has been assumed that while the assembly pathway of Sindbis virus differs in certain details during the infection process in these divergent hosts, the molecular details of assembly result in functionally equivalent virus structures (49). Indeed, Sindbis virus is a chimeric structure: the protein and the RNA are specified by the viral genome, while the nature of the lipid in the virus is determined largely by the host cell.Recent genetic analysis has further demonstrated the chimeric nature of the alphaviruses at the molecular level and has reemphasized the importance of the host-derived membrane as a structural component of the virus particle. Deletions in the protein domain that engages the membrane result in changes in the infectivity of the virus, while having a lesser effect on the process of assembly. The deletions also affect the ability of the virus to assemble in the insect and mammalian hosts (17, 18, 56). These data suggest that the composition of the host cell membrane plays a critical role in the assembly of the virus. The amount of cholesterol in the membrane has been suggested to be critical for both the assembly of virus and the ability of the virus to infect cells (25, 31, 34, 43). To further test this theory, an investigation into the effect of the amount of cholesterol incorporated into the viral membrane on virus infection of and virus production from an insect or mammalian host was warranted.Our model for how these truncated proteins retain the ability to assemble into infectious particles in insect membranes invokes membrane thickness as a primary factor in the stability of shortened membrane anchor domains in the lipid bilayer. This hypothesis was put forth because of the evidence that the high cholesterol content found in mammalian cells can increase membrane thickness and alter physical properties, such as ion permeability and viscosity (36). Cholesterol in the eukaryotic membrane enhances acyl chain packing of the phospholipids, increases mechanical strength, and reduces permeability (57). Insects cannot synthesize cholesterol de novo and depend on dietary cholesterol for their physiological requirements (27). As cholesterol auxotrophs, insect cells in culture can withstand a significant level of cholesterol depletion (47); however, in the presence of serum, they will incorporate cholesterol (37). In the present study, we have used wild-type Sindbis virus specifically to investigate the ability of this virus to incorporate cholesterol into the viral membrane when grown under standard conditions and under conditions of lipid depletion. To address this question, Sindbis virus was grown in BHK or mosquito U4.4, C7-10, or C6/36 cells under conditions of high levels of free lipid and various conditions of lipid depletion and in an additional insect cell line, Sf21, grown under serum-free conditions.     

An Air Drying Technique for Maize Chromosomes without Enzymatic Maceration

The air drying technique, widely used in animal cytogenetics, was adapted for use with Zea mays L. chromosomes. Using a simple protocol without enzymatic maceration and avoiding the inconvenience of the squashing technique, good staining and C-banding were obtained from maize chromosome preparations.     

In Vitro Deoxynucleotidylation of the Terminal Protein of Streptomyces Linear Chromosomes

Single-stranded gaps at the 3′ ends of Streptomyces linear replicons are patched by DNA synthesis primed by terminal proteins (TP) during replication. We devised an in vitro system that specifically incorporated dCMP, the first nucleotide at the 5′ ends, onto a threonine residue of the TP of Streptomyces coelicolor.     

The Dynamic Behavior of Individual Microtubules Associated with Chromosomes In Vitro

Mitotic movements of chromosomes are usually coupled to the elongation and shortening of the microtubules to which they are bound. The lengths of kinetochore-associated microtubules change by incorporation or loss of tubulin subunits, principally at their chromosome-bound ends. We have reproduced aspects of this phenomenon in vitro, using a real-time assay that displays directly the movements of individual chromosome-associated microtubules as they elongate and shorten. Chromosomes isolated from cultured Chinese hamster ovary cells were adhered to coverslips and then allowed to bind labeled microtubules. In the presence of tubulin and GTP, these microtubules could grow at their chromosome-bound ends, causing the labeled segments to move away from the chromosomes, even in the absence of ATP. Sometimes a microtubule would switch to shortening, causing the direction of movement to change abruptly. The link between a microtubule and a chromosome was mechanically strong; 15 pN of tension was generally insufficient to detach a microtubule, even though it could add subunits at the kinetochore–microtubule junction. The behavior of the microtubules in vitro was regulated by the chromosomes to which they were bound; the frequency of transitions from polymerization to depolymerization was decreased, and the speed of depolymerization-coupled movement toward chromosomes was only one-fifth the rate of shortening for microtubules free in solution. Our results are consistent with a model in which each microtubule interacts with an increasing number of chromosome-associated binding sites as it approaches the kinetochore.     

An Effective Method for Selecting siRNA Target Sequences in Mammalian Cells

RNA interference is a gene-silencing phenomenon triggered by dsRNA (double-stranded RNA) and has been widely used for studying gene functions. The short interfering RNA (siRNA) responsible for RNA interference, however, varies markedly in its gene-silencing efficacy. Because this efficacy depends on the selected target sequences, we developed an effective selection method based on the gene degradation measure (priority score) defined by positional features of individual nucleotides. We tested this method experimentally by using it to select new siRNA target sequences in the homo sapiens cyclin B1 gene (CCNB1) and confirmed that it selected highly effective gene-silencing sequences. The proposed method will therefore be useful for selecting new siRNA target sequences in mammalian cells.     

An Improved Squash Technique for Human Male Meiotic Chromosomes: Softening and Concentration of Cells; Mounting in Hoyer's Medium

Human testicular material obtained from autopsies was processed as follows: (1) swelled with 0.3% sodium citrate 1-6 hr; (2) softened with 3 M glucono-delta-lactone 2 hr; (3) stained with aceto- or propiono-carmine overnight; (4) washed in 70% alcohol; (5) macerated in 1:1 alcohol-acetic acid; (6) filtered through gauze to remove tubules and connective tissue; (7) filtrate centrifuged to separate spermatocytes from debris; (8) a drop of supernate from just above the precipitate, containing stained cells, mixed with Hoyer's medium; (9) cover slip applied, preparation warmed, and (10) squashed. Chromosomes remained in good condition for 8 mo.