Phase imaging of moving DNA molecules and DNA molecules replicated in the atomic force microscope.

Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution. In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM. Higher scan rates make it possible to image faster processes. At lower forces the molecules are imaged more gently. Moving DNA molecules are also resolved more clearly in phase images than in height images. Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface. Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.

Properties of biomolecules measured from atomic force microscope images: a review.

AFM images can be used to obtain quantitative or qualitative information about the properties of biomaterials. Examples presented here are: (1) Persistence length measurements of moving and stationary DNA molecules. (2) Force mapping to measure properties such as the elasticity of cells and vesicles. (3) Phase mode imaging to detect variations in materials and properties of the sample surface. (4) Imaging of surfaces at different constant forces.

A regulatory role for the 5' and 3' untranslated regions in differential expression of hsp83 in Leishmania.

Exposure of Leishmania promastigotes to temperatures typical of mammals result in a stress response, which is accompanied by an increase in the steady state level of heat shock transcripts and their translation. Accumulation of the heat shock protein (hsp83) mRNA occurs due to differential decay rates at the altered temperatures, while transcription is unaffected. A similar pattern of post-transcriptional regulation was observed for a transfected chloramphenicol acetyltransferase (CAT) gene, which was flanked at both ends by intergenic regions (IR) of hsp83. Shortening the 5' untranslated region (UTR) by 100 nts produced an active CAT enzyme, but abolished the temperature-dependent regulation of the CAT-hsp83 mRNA turn-over. The 3' UTR is also involved in the temperature-dependent degradation of hsp83 mRNA, since exchange of the hsp83 3' UTR with a parallel fragment from a non-heat shock gene abolished the differential turn-over of CAT mRNA. Thus, the regulated decay of hsp83 mRNA is controlled by sequence or conformational elements present in both upstream and downstream UTRs. Like the endogenous hsp83, translation of CAT mRNA which contained hsp83 UTRs was higher at 35 degrees C. This was observed only with transcripts in which stability increased at elevated temperatures. Modifications which abolished the temperature dependence of CAT mRNA decay, eliminated its elevated translation at the higher temperatures. The correlation suggests a mechanistic link between the translational machinery and mRNA stability.

Expression of heat shock protein 83 in Leishmania is regulated post-transcriptionally.

Mechanisms for regulation of heat shock protein (hsp) 83 expression were examined in Leishmania amazonesis. Transcripts of hsp83 accumulated upon temperature elevation; however, in contrast to non-protozoan eukaryotes (i.e. Drosophila, yeast, avian or human cells), no transcriptional activation was observed. The increase in the hsp83 mRNA level evolved from temperature induced variations in mRNA turn-over: the hsp83 transcript was rapidly degraded at normal temperatures, whereas heat shock led to its stabilization. The quick decay of the mRNA at lower temperatures was dependent on active protein synthesis. A similar pattern of regulation was observed for the transfected chloramphenicol acetyltransferase (CAT) gene, which was flanked by sequences from the hsp83 intergenic region (IR), and cloned into the pX transfection vector (pX-ICI). CAT mRNA was abundant at normal temperatures and further accumulated upon temperature elevation. The altered turn-over rates of CAT mRNA at the different temperatures were observed only in the presence of flanking hsp83 IR sequences. The increase in temperature also affected translational regulation of hsps, and synthesis of hsp83 was more efficient at 35 degrees C than at 26 degrees C. However, the effect of translation was transient, and the steady state level of the protein was hardly altered.

Intergenic sequences from the heat-shock protein 83-encoding gene cluster in Leishmania mexicana amazonensis promote and regulate reporter gene expression in transfected parasites.

Regulation of expression from hsp83 gene cluster encoding heat-shock protein (HSP) 83 of the protozoan parasite Leishmania mexicana amazonensis (L.m.a) was examined. The first gene from this cluster, along with 8 kb of flanking sequences, was cloned, and intergenic region (IR) sequences were found upstream from the cluster. L.m.a. parasites were electroporated with a plasmid (pICI) in which the chloramphenicol acetyltransferase (CAT)-encoding gene (cat) was cloned between two IRs derived from an internal repeat unit of the hsp83 cluster, resulting in CAT activity at 26 degrees C. Exposure of cells transfected with this plasmid to a 35 degrees C heat shock led to an increase in CAT activity, within a range similar to that observed for the accumulation of hsp83 steady-state mRNA at 35 degrees C. S1 analysis of the hsp83 mRNA showed that the major part of the IR was transcribed and mostly present as 3' non-translated extensions. Deletion analysis of the flanking regions indicated that the presence of IR sequences, both upstream and downstream from cat, was critical to its expression. Partial deletions that removed the original AG splice acceptor site (leaving 289 bp upstream) and downstream IR sequences (leaving 200 bp) did not eliminate CAT activity. However, this combined deletion altered the effect of temperature on cat expression in transfected cells, as compared with the activity measured in cells transfected with the original plasmid.

Induction of protein-tyrosine-phosphatase activity by interleukin 6 in M1 myeloblastic cells and analysis of possible counteractions by the BCR-ABL oncogene.

Interleukin 6 (IL-6) induces in M1 myeloblastic cells growth arrest and terminal differentiation toward monocytes. It is reported here that IL-6 reduced by 5- to 20-fold the tyrosine phosphorylation of cellular proteins in these cells. The same-fold reduction was also observed in M1 cells that were transfected with the BCR-ABL deregulated protein kinase. In these stable clones, the levels of tyrosine phosphorylation of cellular proteins were 30- to 100-fold higher than in the parental cells. IL-6 did not reduce the expression levels or the inherent tyrosine kinase activity of BCR-ABL p210. By measuring the protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in crude cell lysates, we found that protein dephosphorylation resulted, at least partially, from induction of PTPase activity by IL-6. The induction of PTPase in the BCR-ABL-transfected clones was not sufficient to confer the minimal protein phosphorylation levels characteristic of IL-6-treated cells. Yet, the transfected M1 clones showed normal growth and differentiation responses to IL-6. None of the gene responses to IL-6 including suppression in the levels of c-myc, c-myb, and cyclin A mRNA; junB and c-jun mRNA induction; and dephosphorylation of retinoblastoma protein were rescued by the BCR-ABL oncogene. The functional relevance of PTPase induction by IL-6 is discussed.

Otitis externa - bacteriological survey.

Otitis externa is one of the most common problems faced by the otolaryngologist, and in some clinics constitutes up to 40% of patients. Although not lethal, it may be a most debilitating disease. The external ear is an epithelium-lined cul-de-sac with many sweat and cerumeniferous glands whose secretions are an excellent medium for bacterial growth. Bacterial surveys done in the USA and in Israel 30 years ago proved Staphylococcus aureus to be the major pathogen. During the years the major pathogen changed, and in recent surveys Pseudomonas aeruginosa was found to be the dominant pathogenic bacterium. The purpose of this article is to present the results of a bacteriological survey done in Israel on patients suffering from otitis externa in the years 1979-1980. A discussion is presented with regard to the meaning of the review. We tried to establish whether a certain factor could be considered to be the cause of otitis externa.

Studies on the antimicrobial activity of amniotic fluid.

Sixty-one amniotic fluid samples from women in their second and third trimesters of pregnancy were examined for antimicrobial activity. Seventy per cent of the fluids were found to be active. The factor or factors responsible for this activity were present in low concentrations. The presence of spermine in the fluids accounted for some of the antimicrobial activity.

Heteroimmunization to the capsular polysaccharide of Haemophilus influenzae type b induced by enteric cross-reacting bacteria.

Cross-reacting Escherichia coli strains Easter and 89 and Bacillus pumilis fed to newborn rabbits and E. coli fed to adult rhesus monkeys did not exert untoward reactions. The E. coli regularly colonized the newborns' intestinal tract from 1 to 7 weeks. High doses of E. coli were necessary to colonize adult primates. Colonization occurred in fewer newborn rabbits and lasted only 1 to 3 weeks with B. pumilis. Colonized newborn rabbits and adult rhesus had an active Haemophilus influenzae type b (HITB) immune response. In the rabbit, colonization resulted in accelerated induction of immunoglobulin (Ig) M-. IgA-, and IgG-producing cells in the spleen, mesenteric lymph nodes, and Peyer's patches after HITB challenge. E. coli-fed and control newborn primates were naturally colonized with nasopharyngeal and enteric cross-reacting bacteria and both groups rapidly developed HITB antibodies in the absence of the homologous organisms. Human newborn stool cultures, taken at the time of discharge from the nursery, showed a 0.9% carriage rate for cross-reacting E. coli. These "carrier" infants acquired HITB antibodies more rapidly than their age-matched "noncarrier" controls.

Enteric bacteria cross-reactive with Neisseria meningitidis groups A and C and Diplococcus pneumoniae types I and 3.

Enteric bacteria of 1,335 individual strains were studied for serological cross-reactions with Neisseria meningitidis groups A and C and Diplococcus pneumoniae types I and III. Enterobacterial antigens cross-reactive with the capsular polysaccharides of these four bacteria were found. Bacteria cross-reactive with noncapsular antigens of meningococci and pneumococci were also observed. Since some enteric bacteria possess antigens with serological specificities similar to those of meningococci, the possibility that enteric bacteria cross-reactive with meningococcal antigens provide an antigenic stimulus for the observed age-related "natural" immunity to this pathogen is discussed.