Imaging techniques in microbiology

Recent advances in optical imaging have dramatically expanded the capabilities of the light microscope and its usefulness in microbiology research. Some of these advances include improved fluorescent probes, better cameras, new techniques such as confocal and deconvolution microscopy, and the use of computers in imaging and image analysis. These new technologies have now been applied to microbiological problems with resounding success.     

Relationship between hypertension, hypertrophy, and opening angle of zero-stress state of arteries following aortic constriction

Examination of changes occurring in the zero-stress state of an organ provides a way to study cellular growth in the organ due to change of physical stresses. The zero-stress state of the aorta is not a tube. It is a sector with an opening angle that varies with the location on the aorta and changes with cellular remodeling. Blood vessel remodeling can be induced by imposing a constriction on the abdominal aorta by a metal clip (aortic banding), which causes an increase of blood pressure, hypertrophy of the aortic wall, and large change of opening angle. The correlation of the opening angle with the blood vessel wall thickness and blood pressure changes in rat's aorta due to aortic banding is presented in this report. The opening angle changes daily following the aortic banding. Blood pressure rises in vessels of the upper body, but that in the lower body decreases at first and then rises to an asymptotic value. Blood vessel wall thickness increases in rough proportion to blood pressure. Vessel diameter changes also. But the most dramatic is the course of change of the zero-stress state. Typically, the time to reach 50 percent of asymptotic hypertrophy of blood vessel wall thickness is about 3-5 days. The corresponding time for blood pressure is about 7 days. The opening angle of the zero-stress state, however, increases rapidly at first, reaches a peak in about 2 to 4 days, then decreases gradually to a reduced asymptote. The exact values of the time constants depend on the location along the aortic tree. In general, the course of change of residual strain is very different from those of the blood pressure and the blood vessel wall thickness.     

Heme maintains catalytically active structure of cytochrome P-450

Treatment of purified cytochrome P-450 LM2 and its liposome-bound form with hydrogen peroxide led to complete destruction of the P-450 heme. The apoenzyme thus produced could be reconstituted to catalytically active cytochrome P-450 by incubation with hemin, the reconstitution efficiency being 50% for the soluble enzyme and 80% for the liposome-bound enzyme. The removal of heme from the soluble hemoprotein resulted in a 3-fold decrease in the efficiency of its incorporation into sonicated liposomes. The contents of 5 secondary structure forms in the native, apoand reconstituted holoenzymes were estimated from their circular dichroism spectra. It was thus found that the helix content increased from 34% to 60% upon removal of the heme from the native enzyme. We suggest that the increase in the helix content leads to a reduction of the incorporation efficiency into liposomal membranes.     

Evaluation of neuron-based sensing with the neurotransmitter serotonin

Results are presented on the development of a novel biosensor which will use neurons or neuronal components as both the recognition elements and primary transducers for analyte quantitation. This concept is demonstrated and evaluated by exposing identified neurons from the visceral ganglia of the pond snail Limnea stagnalis to the model analyte serotonin. Experiments reveal a reversible, concentration-dependent increase in the rate of spontaneous action potential generation, over a concentration range of four orders of magnitude. Studies with the antagonist methysergide verify that this response is mediated through serotonin-sensitive receptors. Exposure of the neurons to serotonin causes the firing frequency to rapidly increase to a maximum and then slowly diminish to a sub-optimal level. It was found that the maximum frequency provides an indication of chemical concentration that is repeatable. Data are also presented which further advanced the field of neuronal biosensing by demonstrating both the effects of cell to cell variability on response reproducibility and the effects of the desensitizing response on the operation of a neuron-based sensor in both a continuous and discontinuous mode.     

Changes of zero-stress state of rat pulmonary arteries in hypoxic hypertension

Zero-stress state of the main pulmonary arteries, from the main trunk to a vessel with a lumen diameter approximately 60 microns, was determined in 25 normal control and 38 hypoxic pulmonary hypertensive rats. Pulmonary hypertension was induced by placing the rats in a hypoxic chamber with 10% O2-90% N2 at atmospheric pressure. The zero-stress state of each vessel was obtained by first cutting the vessel transversely into a series of rings and then cutting each ring radially, whereupon the ring opened into a sector, which is characterized by an opening angle defined as the angle subtended between two lines originating from the midpoint of the inner wall (endothelium) to the tips of the inner wall. Whereas the pulmonary blood pressure increased monotonically during the development of pulmonary hypertension, the opening angle followed a different course; e.g., the values (means +/- SD) of the opening angle at the pulmonary trunk at times 0 (control) and 2, 12, 28, 96, 144, 240, 480, and 720 h after exposure to hypoxia are, respectively, 294 +/- 30 degrees, 378 +/- 24 degrees, 385 +/- 12 degrees, 374 +/- 11 degrees, 246 +/- 63 degrees, 267 +/- 49 degrees, 193 +/- 19 degrees, 195 +/- 83 degrees, and 239 +/- 38 degrees. Trends at other places on the artery are similar, but the magnitudes differ. In this period of time, intimal edema and thickening were found. The intima media thickened rapidly from 48 to 240 h and then more slowly from 240 to 720 h. Adventitia thickened later; its thickness exceeded that of the intima media at approximately 96 h. Thus the changes of zero-stress state of the pulmonary arteries are seen to be related to the nonuniform remodeling of the vessel wall as revealed by the edema, blebs, and thickening of different layers.     

Dynamic light scattering investigations of human erythrocyte spectrin.

Dynamic light scattering measurements were performed on spectrin from human erythrocytes in 25 mM Tris buffer at pH 7.6 with 100 mM NaCl and 5 mM EDTA. Measurements were made on spectrin solutions prepared as dimers and tetramers over the temperature range from 23 to 41 degrees C, as a function of the square of the scattering vector (K2) over the range of 0.7 x 10(10) cm-2 less than or equal to K1 less than or equal to 20 x 10(10) cm-2. Analysis of the autocorrelation functions collected for these solutions revealed the presence of two predominant motional components over the entire range of K2. Plots of the diffusion coefficients (D20) of these components, with viscosity and temperature corrected to water at 20 degrees C, as a function of K2 indicated three rather distinct regions, flat regions at low and high K2 joined by a sloping intermediate region. At small K2 (less than or equal to 4 x 10(10) cm-2) the D20 values were (7.3 +/- 2.0) x 10(-8) cm2/s for the slow component and (20.3 +/- 2.0) x 10(-8) cm2/s for the fast component. At large K2 (greater than or equal to 10 x 10(10) cm-2) the values increased to (13.0 +/- 2.0) x 10(-8) cm2/s for the slow component and (39.4 +/- 2.0) x 10(-8) cm2/s for the fast component. In the intermediate K2 region, D20 is a linear function of K2 and appears as a transition between the low and high K2 regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation and characterization of monoclonal antibodies to the putative CD4-binding domain of human immunodeficiency virus type 1 gp120.

A panel of seven monoclonal antibodies against the relatively conserved CD4-binding domain on human immunodeficiency virus type 1 (HIV-1) gp120 was generated by immunizing mice with purified gp120. These monoclonal antibodies reacted specifically with gp120 in an enzyme-linked immunosorbent assay and Western blots (immunoblots). By using synthetic peptides as antigens in the immunosorbent assay, the epitopes of these seven monoclonal antibodies were mapped to amino acid residues 423 to 437 of gp120. Further studies with radioimmunoprecipitation assays showed that they cross-reacted with both gp120 and gp160 of diverse HIV-1 isolates (HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, and HTLV-IIIWMJ). They also bound specifically to H9 cells infected with HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, HTLV-IIIZ84, and HTLV-IIIZ34 in indirect immunofluorescence studies. In addition, they blocked effectively the binding of HIV-1 to CD4+ C8166 cells. Despite the similarity of these properties, the monoclonal antibodies differed in neutralizing activity against HTLV-IIIB, HTLV-IIIRF, and HTLV-IIIAL, as demonstrated in both syncytium-forming assays and infectivity assays. Our findings suggest that these group-specific monoclonal antibodies to the putative CD4-binding domain on gp120 are potential candidates for development of therapeutic agents against acquired immunodeficiency disease syndrome.     

Effect of intermittent exposure and drug-free intervals on the in vitro vascular tolerance to nitroglycerin

Recent reports suggest that intermittent nitroglycerin (NTG) treatment, incorporating a daily nitrate-free interval, can avoid the tolerance associated with continuous NTG therapy. This study has investigated whether an in vitro model of NTG tolerance could be used to examine the mechanisms of tolerance avoidance (by intermittent NTG exposure) and tolerance reversal. Isolated rat abdominal aortic rings were exposed to 55 microM NTG at varying intervals over a 60 min period, and the concentration-relaxation curves to NTG were subsequently determined. Intermittent NTG exposure (either 12 x 0.5 min or 3 x 2 min) significantly reduced NTG tolerance compared to continuous exposure over the same period of time (60 min). The diminished tolerance was apparently due to the reduced total exposure time, since the NTG responsiveness of aortic rings exposed to NTG intermittently or continuously for 6 min of the incubation period was not significantly different. Under the present conditions, in vitro NTG tolerance could be reversed if sufficient washout time was allowed. Thus, aortic rings exposed to NTG for the initial, but not the final, 6 min of incubation were not tolerant to NTG. In addition, rings exposed to NTG for 3 min exhibited near-maximal tolerance after 5 min washout, but no tolerance after 60 or 120 min washout. It appears, therefore, that the isolated vessel retains the "repair" mechanism responsible for tolerance reversal under the present conditions. This study suggests that the in vitro model of NTG tolerance may be useful for investigating the characteristics and mechanisms of tolerance avoidance and reversal, as well as tolerance induction.     

Synthesis and secretion of hepatitis B middle surface antigen by the methylotrophic yeast Hansenula polymorpha

The methylotrophic yeast, Hansenula polymorpha, has been developed as a host system for the synthesis of heterologous proteins. The middle surface antigen of hepatitis B virus (preS2-HBsAg) has been synthesized under the control of a methanol-regulated promoter derived from the methanol oxidase-encoding gene. The synthesized preS2-HBsAg protein was found to be secreted outside the cell membrane into the periplasm and further excreted into the culture medium following permeabilization of the cell wall with beta-1,3-glucanase (beta Glu). Cell cultures treated with beta Glu were able to continuously synthesize and secrete 22-nm particles of preS2-HBsAg into the medium for several days. The overall yield of antigen from treated cultures was found to be over threefold greater than that of untreated controls. The observation that complex supramolecular structures, such as the 22-nm particles of preS2-HBsAg, can be secreted by H. polymorpha and released into the medium, suggests the potential for these yeasts to be an alternative secretory host.