Search PubMed⌕ Search

Biomedical subjects

J M Mackenzie

Publications and source records attributed to J M Mackenzie.

At least 37 records · Page 2Linked to original sources
PubMed1996

Bleb formation and F-actin distribution during mitosis and tumor necrosis factor-induced apoptosis.

The murine cell line C3HA has been used extensively in studies of the cytopathology that accompanies TNF-induced cytolysis. This cell line undergoes an apoptic form of cell death characterized by plasma membrane blebbing and cytoplasmic boiling. Since plasma membrane blebs also appear on C3HA cells during mitosis, in this report we have compared these blebs with those that appear during apoptosis to determine whether they represent related structures. Our results reveal several differences. During mitosis, the blebs that appear are smaller and more heterogeneous in size than are those that appear during apoptosis. In addition, during mitosis bleb formation is preceded by the appearance of microvilli on the cell surface. No microvilli are observed during apoptosis. The staining pattern with rhodamine phalloidin also differed between mitotic and apoptic blebs, indicating a difference in their content of f-actin. The blebs that form during mitosis stained in a bright, uniform manner, suggesting an association with f-actin. In contrast, apoptic blebs were stained only at their base, the bleb itself being devoid of f-actin-associated staining. This difference may help explain why mitotic blebs are reintegrated into the cell surface, while the blebs that form during apoptosis are not.

Actins↗
PubMed1996

Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication.

Although many workers have investigated the maturation and processing of the flavivirus nonstructural glycoprotein NS1 in infected cells, these studies have provided little insight into a possible function for NS1. In this study we investigated the subcellular localization of NS1 both by immunofluorescence and cryo-immuno electron microscopy of infected Vero and C6/36 cells. NS1 was found to be tightly associated with intracellular membranes, in particular with vesicle packets and large cytoplasmic vacuoles. Surprisingly, NS1 did not associate with mature virus particles, a finding that is inconsistent with the postulated role for this protein in virion assembly and/or maturation. However, dual-labeling experiments did reveal in both the confocal immunofluorescence and cryo-immuno EM studies the colocalization of NS1 with the viral dsRNA replicative form. Furthermore, localization of the dsRNA to the vesicle packets and cytoplasmic vacuoles seen in infected Vero and C6/36 cells, respectively, suggests that these structures may comprise the flavivirus replication complex. These findings provide further insights into the maturation pathway of NS1 and suggest a possible role for this protein in viral RNA replication.

Animals↗
PubMed1996

Improved membrane preservation of flavivirus-infected cells with cryosectioning.

Ultra-cryomicrotomy and electron microscopy were used to investigate membranous structures in dengue virus-infected mammalian and insect cells. The cryo-sectioned samples displayed ultrastructure comparable to their resin-embedded counterparts with all previously identified virus-induced structures being observed. Structures not previously identified were also found. In particular, membrane-bound packets of vesicles, 100-200 nm in diameter were seen distributed throughout areas of virus-induced membrane proliferation. These packets were clearly distinct from virion arrays. Small smooth membrane vesicles, previously found to contain thread-like enclosures (M.L. Ng, J. Gen. Virol. 68 (1987) 577-582), were frequently observed to contain dense staining material, however the exact nature of this material remains unclear. Virus-induced modification of golgi-like and/or ER membranes was also observed and may represent early events in the generation of the smooth membrane vesicles seen during infection. We suggest that cryosectioning is the method of choice to investigate membrane rearrangement induced by this family of viruses and that a diamond knife and modified staining techniques, as utilised in this report, be employed to enhance morphology and section preservation.

Animals↗
PubMed1992

Binding of Lactobacillus reuteri to fibronectin immobilized on glass beads.

Human fibronectin was immobilized on glass beads. The beads were used to evaluate binding of Lactobacillus reuteri to fibronectin. Organisms bound to the glass beads were detected using fluorescence microscopy after treatment with acridine orange. This binding was confirmed and quantified with the use of [3H]-labelled organisms. Three strains of Lactobacillus reuteri, three strains of Lactobacillus acidophilus and one strain of Lactobacillus fermentum were tested for binding capacity. L. reuteri strain 1063 exhibited a strong binding to the immobilized fibronectin, and L. acidophilus 1754 showed a slight binding. The binding of L. reuteri to the fibronectin was mediated by a protein as judged by the absence of binding after treatment of the bacteria with proteolytic enzymes. Treatment of the bacteria with urea, SDS and heat (80 degrees C) also reduced binding. Treatment of the bacterial cells prior to the assay with fibronectin interfered with binding. Albumin did not show this interaction.

Bacterial Adhesion↗
PubMed1990

Isolation, identification, and characterization of a feather-degrading bacterium.

A feather-degrading culture was enriched with isolates from a poultry waste digestor and adapted to grow with feathers as its primary source of carbon, sulfur, and energy. Subsequently, a feather-hydrolytic, endospore-forming, motile, rod-shaped bacterium was isolated from the feather-degrading culture. The organism was Gram stain variable and catalase positive and demonstrated facultative growth at thermophilic temperatures. The optimum rate of growth in nutrient broth occurred at 45 to 50 degrees C and at pH 7.5. Electron microscopy of the isolate showed internal crystals. The microorganism was identified as Bacillus licheniformis PWD-1. Growth on hammer-milled-feather medium of various substrate concentrations was determined by plate colony count. Maximum growth (approximately 10 cells per ml) at 50 degrees C occurred 5 days postinoculation on 1% feather substrate. Feather hydrolysis was evidenced as free amino acids produced in the medium. The most efficient conditions for feather fermentation occurred during the incubation of 1 part feathers to 2 parts B. licheniformis PWD-1 culture (10 cells per ml) for 6 days at 50 degrees C. These data indicate a potential biotechnique for degradation and utilization of feather keratin.

Journal Article↗
PubMed1990

A conformational change in Sindbis virus glycoproteins E1 and E2 is detected at the plasma membrane as a consequence of early virus-cell interaction.

A conformational change in the structure of Sindbis (SB) virus was detected after virion attachment to baby hamster kidney cells but before internalization. The alteration was manifested as increased virion binding of certain glycoprotein E1 and E2 monoclonal antibodies (MAbs) that recognized transitional epitopes. These epitopes were inaccessible to MAb on native virions but became accessible to their cognate MAbs in the early stages of infection. Transit of virions through a low-pH compartment apparently was not required for the conformational change. Exposure of transitional epitopes was unaffected by treatment of BHK cells with NH4Cl and occurred normally in Chinese hamster ovary cells temperature sensitive for endosomal acidification. However, the rearrangement was correlated with both the time course and temperature dependence of SB virus penetration, and the rearrangement occurred earlier with an SB virus mutant having an accelerated penetration phenotype. In addition, MAb to a transitional epitope, a probe specific for rearranged particles, retarded penetration of infectious virions. These results suggested that the SB virus E1/E2 glycoprotein spike undergoes a structural rearrangement as a consequence of virion interaction with the cell surface and that this altered virion form may be an important early intermediate in an entry pathway leading to productive infection.

Ammonium Chloride↗
PubMed1988

Antibody-mediated activation of Sindbis virus.

The biological activity of an anti-Sindbis monoclonal antibody (MCAB 49) has been explored. The antibody recognizes an epitope on the E2 glycoprotein of Sindbis virus and, in the presence of complement (C'), neutralizes virus infectivity. In the absence of C', reaction of the antibody with our laboratory strain of Sindbis, SB, increased the number of plaque-forming units (PFU) detected on baby hamster kidney (BHK) cells rather than neutralizing virus infectivity. The elevated titers of SB approached, but never exceeded, the number of virions calculated from the particle:PFU ratio, indicating that the additional PFU might have resulted from activation of normally noninfectious particles. The apparent activation could not be attributed to disaggregation of SB by MCAB 49 as shown by ultraviolet inactivation experiments with antibody-treated and untreated virus preparations. Fc receptors did not appear to be involved in the antibody-mediated activation. Fab' and F(ab')2 fragments of MCAB 49 also increased the number of observed PFU of SB. Control monoclonal antibodies of the same isotype, but specific for the tobacco etch virus capsid protein, were unable to compete for cellular binding sites with the SB/MCAB 49 complex. Rather, the SB/MCAB 49 complex appeared to utilize the same receptor(s) as SB in that SB and the SB/MCAB 49 complexes competed with each other for binding sites on BHK cells. Binding studies with 32P-labeled SB showed that a higher proportion of MCAB 49 activated virions than untreated virions associated with BHK cells. Moreover, activated virions were much less susceptible to elution. These results suggest that reaction which MCAB 49 may facilitate successful attachment of SB to its receptor, or receptors, on BHK cells.

Animals↗
PubMed1988

Fatal pneumonia with terminal emaciation in nude mice caused by pneumonia virus of mice.

Athymic nude mice used as sentinel animals in a mouse holding room died of pneumonia 17 to 32 weeks after being placed in the room. Lesions in the pulmonary parenchyma consisted of monocytic exudate, epithelial cell necrosis, hemorrhage, fibrin deposition and interstitial fibrosis. Septal edema, septal cell necrosis and septal capillary stasis were common, but there was limited sloughing of bronchial lining epithelium. Indirect fluorescence microscopy (IFA) of lung sections using pneumonia virus of mice (PVM) antibody was positive. The pneumonia and IFA results were reproduced in euthymic mice inoculated experimentally with lung suspension from naturally infected mice or with tissue culture fluid from cultures infected with American Type Culture Collection PVM. The lungs of a naturally infected nude mouse were studied by transmission electron microscopy. Virus growth was found on Type II alveolar epithelium and on poorly differentiated replacement alveolar epithelium. Virus particles appeared as long exophytic filaments containing one to six linearly arranged nucleocapsids. Inclusion bodies and intracellular virus structures were not observed.

Animals↗
PubMed1986

Assembly of overlapping DNA sequences by a program written in BASIC for 64K CP/M and MS-DOS IBM-compatible microcomputers.

The SEQALIGN programs1 described in this report aid in the assembly of up to 100 individual overlapping DNA sequences generated by M-13 subcloning and sequencing methods. The program produces a printout of the aligned sequences presented in register. Use of the program will be facilitated because 1) it is written with the Microsoft BASIC interpreter, 2) sequence data may be entered and edited using WORDSTAR or similar word processing programs, and 3) hardware requirements for execution of the program on CP/M or MS-DOS (IBM-PC compatible) systems are minimal.

Base Sequence↗
PubMed1984

Birefringent periodicities in amphibian rod outer segments.

Birefringence variations, seen as regularly spaced altering light and dark rings (bands), have been observed along the length of unfixed, freshly isolated rod outer segments (ROS) of both Rana pipiens and Xenopus laevis. In our hands, the spatial frequency of the banding pattern is from 1.3-1.6u/band in Rana pipiens and 1.8-2.2u/band in Xenopus laevis ROS, both corresponding closely to determinations we made in the same animals of the quantity of new ROS disks added each day. To further probe this correlation, Xenopus laevis were maintained at 16 degrees C to lower the disk renewal rate. A similar correspondence was found, with the banding pattern and renewal rate both at 0.8-1.Ou/day. Further experiments involving Xenopus laevis placed on altered lighting cycles have suggested the existence of two normally superimposed periodicities. The more intense component is driven by the environmental light cycle, and thus may be regarded as diurnal. The less intense component is seen infrequently, suggesting lability, and apparently follows a 24-hour period in both constant light and darkness.

Animals↗
PubMed1983

Transition in the thin-filament arrangement in rat skeletal muscle.

The transition in thin-filament arrangement from tetragonal near the Z-band to trigonal in the A-band was investigated by computer-assisted analysis of thin-filament arrangement in serially cross-sectioned rat muscle. Extensor digitorum longus (EDL; fast) muscle from adult rats and adult, 9-day and 3-day neonatal soleus (slow) muscle were serially cross-sectioned from the H-zone of one sarcomere to the H-zone of an adjacent sarcomere. Thin-filament arrangement was analysed throughout the I-band, and particularly at three levels of the sarcomere: in the I-band, one section (0.06 microns) from the Z-band; six sections (0.36 microns) from the Z-band; in the A-band, two sections from the A-I junction (0.72 microns from the Z-band). Data for radical distributions and annular distributions were obtained by computer. In all muscles studies, thin-filament arrangement exhibited four-neighbour ordering throughout the I-band. Thin-filament arrangement exhibited three-neighbour ordering only in the A-band. The transition in thin-filament arrangement from four-neighbour to three-neighbour ordering occurred within 0.12 microns of the A-I junction in muscles fixed at rest length. In adult soleus that had been stretched 20% so that the A-I junction moved away from the N2-line, the transition in thin-filament arrangement occurred in the I-band within a region 0.4-0.5 microns from the Z-band. This region corresponds to the N2-line region of the I-band. Thus, the transition from four-neighbour to three-neighbour ordering occurs in the I-band independent of the thin filament-thick filament interaction. We conclude that some inherent feature of the I-band or thin filament-thin filament interaction imposes a four-neighbour ordering on thin filaments from the Z-band to the N2-line.

Animals↗
PubMed1978

Muscle development in Caenorhabditis elegans: mutants exhibiting retarded sarcomere construction.

We have studied the structural changes within the body-wall muscle cells of Caenorhabditis elegans during postmitotic development. In wildtype, the number of sarcomeres progressively increases, and each sarcomere appears to grow in length and depth continuously during this period. In mature wild-type cells, the anterior-most body-wall muscle cells have 6--7 sarcomeres; the rest have 9--10 sarcomeres per cell. Twelve mutants in the unc-52 II gene exhibit markedly retarded sarcomere construction and progressive paralysis. Several unc-52 mutants, such as the severely paralyzed SU200, produced only 2--3 sarcomeres per body-wall muscle cell, while the other mildly paralyzed unc-52 mutants, such as SU250, build 3--4 sarcomeres per muscle cell. Other structures such as the pharynx and even the noncontractile organelles of the body-wall muscle cells do not appear to be structurally or functionally altered. The unc-52 body-wall sarcomeres become moderately disorganized as they are outstripped by cell growth; sufficient order is preserved, however, so that the majority of thick and thin filaments still interdigitate. The myosin heavy chains of SU200 body-wall muscle fail to accumulate normally, while the pharyngeal myosin heavy chains do not appear to be specifically affected. This biochemical result correlates well with the specificity of morphological changes in the mutant. A model is discussed in which the biochemical and morphological deficits are explained by a simple regulatory mechanism.

Animals↗
PubMed1975

Reversible redistribution of phytochrome within the cell upon conversion to its physiologically active form.

The intracellular localization of phytochrome was seen in dark-grown oat (Avena sativa L., cv. Garry) and rice (Oryza sativa L., cv. unknown) shoots after various light treatments using an indirect peroxidase-antiperoxidase antibody labeling method. Phytochrome is generally distributed throughout the cytoplasm in cells of tissue that had not been exposed to light prior to fixation. Within, at most, 8 min after the onset of saturating red irradiation, phytochrome, now present in the far-red-absorbing form, becomes associated with discrete regions of the cell. These regions do not appear to be nuclei, plastids, or mitochondria. After phototransformation back to the red-absorbing form originally present, phytochrome slowly resumes its general distribution. It is possible that this discrete localization of the far-red-absorbing form of phytochrome represents a physiologically significant binding with a receptor site in the cell.

Cytoplasm↗