The molecular organization of myosin in stress fibers of cultured cells.

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method.

Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system.

Virological and immunological analysis of a triple combination pilot study with loviride, lamivudine and zidovudine in HIV-1-infected patients.

OBJECTIVE: To compare two antiretroviral regiments, loviride plus lamivudine (3TC) plus zidovudine (ZDV) (triple combination) and loviride plus ZDV (double combination) in terms of pharmacokinetic interactions, tolerability, safety, and immunological and virological efficacy. STUDY DESIGN: An open, case-controlled, pharmacokinetic and 24-week continuous treatment pilot study. PATIENTS: Twenty p24 antigen-positive patients, 10 per treatment group, were matched according to p24 antigenaemia less or more than 100 pg, CD4 count less or more than 150 x 10-(6)/l, and gender. Eight out of 10 cases and seven out of 10 controls had received previous antiretroviral therapy. RESULTS: No clinically relevant pharmacokinetic interactions were observed. Both treatment combinations were well tolerated. Median absolute and percentage CD4 count increases above baseline were more pronounced in the triple combination arm than in the double combination arm. Median p24-antigen and plasma viraemia level decreases below baseline were more pronounced in the triple combination arm. The M(184)I/V mutation was detected in all plasma samples of triple combination patients examined at week 12. Mutations conferring resistance to loviride and ZDV were found in a significant subset of patients in both treatment arms. CONCLUSIONS: Both combination regimens have an excellent safety/tolerability profile, but a higher level of in vivo efficacy is achieved by the triple combination, despite genotypic changes conferring resistance to one or all of these agents. The conclusions drawn are limited by small population size and the heterogenous pretreatment history. However, they support the validity of and strongly encourage a rationally designed multidrug combination approach to HIV therapy.

Sequential immunostaining (gold/silver) and complete protein staining (AuroDye) on Western blots.

A double staining method is described which combines immunodetection with sensitive staining of the complete electropherogram on the same membrane. The method is based on the use of Tween 20 as blocking agent, and uses immunogold/silver staining of specific antigens and gold staining of the overall protein pattern with AuroDye. This double staining makes possible the exact location of an immunodetected band within a complex protein pattern.

Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining.

The use of colloidal gold and colloidal gold followed by silver enhancement as marker for dot or blot immune overlays is described. Colloidal gold probes concentrating at the sites of immune reaction gradually develop a pinkish colour during incubation that can be seen with the naked eye. With a physical developer, very high sensitivity and contrast is obtained by silver precipitation on the gold marker. Comparison of the immunogold and immunogold/silver staining methods with the indirect, PAP and ABC immunoperoxidase methods demonstrates that colloidal gold probes are excellent markers for immune overlay assays.

Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations.

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.

Cytochemical profile of immunoregulatory T-lymphocyte subsets defined by monoclonal antibodies.

Immunogold staining in combination with enzyme cytochemistry was used to determine the cytochemical profile of the immunoregulatory T-lymphocyte subpopulations defined by the monoclonal antibodies OKT3, OKT4, OKT8, and OKM1 in normal peripheral blood. Leukocyte suspensions were first incubated with the monoclonal mouse antibodies and then with colloidal gold-labeled goat antimouse antibodies. The cells were fixed and cytocentrifuge preparations were made. Cytochemical reactions for the detection of peroxidase, acid alpha-naphthyl acetate esterase, acid phosphatase, and beta-glucuronidase were performed on these preparations. Under light microscopy, lymphocytes reacting with the monoclonal antibodies had numerous dark granules around their surface membrane. In the cytoplasm the intracellular enzymatic activities were stained. The T-lymphocytes were characterized by a dot-like activity for the three enzymes. No significant difference could be found between the cytochemical profile of the T-helper (OKT4 positive) and T-cytotoxic suppressor cell populations (OKT8 positive). A few cells with lymphocyte morphology reacted with the OKM1 monoclonal antibody. Their cytochemical characteristics were different from those of mature T-cells (OKT3 population) or mature monocytes. From the comparison of their cytochemical characteristics, we can conclude that there is little correlation between the immunoregulatory T-lymphocyte subsets defined by these monoclonal antibodies and those defined by Fc receptors.

Clinical experience with cyclandelate in insulin-dependent diabetic patients with neuropathy.

Previous trials have demonstrated a clinical and electrophysiological improvement of diabetic peripheral polyneuropathy in diabetic patients treated with cyclandelate at a dosage of 1600 mg/day. Hence, a double-blind randomised trial was started in 16 insulin-dependent diabetic patients presenting with symptoms of neuropathy, an increased vibration perception threshold (VPT), disturbed tendon reflexes at lower limbs and an EMG showing a significantly decreased motor nerve conduction velocity (MCV) of the peroneal nerves. The placebo-treated group and the cyclandelate-treated group were not significantly different regarding age, duration of diabetes and level of metabolic control (measured as total HbA1), which remained unchanged during the year of observation. In the cyclandelate-treated group, pathological sensation improved significantly in 7 of 8 patients. MCV, measured under standardised conditions, increased significantly during the first 6 months of treatment, while mean VPT did not change. In the placebo group 3 of 8 patients showed an improvement of sensation, 3 did not feel any change and 2 worsened. Neither mean MCV nor VPT changed significantly. No severe side effects were observed during the study period.

Sequential use of the PAP and immunogold staining method for the light microscopical double staining of tissue antigens.

Double immunoperoxidase staining using different couplers can give various combinations of colours on a single tissue section to achieve a comparable picture of different antigens. However, the colour combinations achieved to date are not entirely satisfactory. A double immunostaining procedure is introduced here, combining the peroxidase anti-peroxidase (PAP) and immunogold staining (IGS) methods. The IGS method is a new, simple, sensitive and reliable approach to immunostaining at the light microscopic level. It was carried out in three ways. Firstly, a two-step method was used in which the second layer was goat anti-rabbit IgG absorbed onto gold particles (GAR/Au20). Secondly, a three-step method was employed where the second layer was unlabelled goat anti-rabbit IgG and the third layer was a rabbit antibody to peroxidase absorbed onto the gold particles (RAP/Au20) and acting as a gold-labelled IgG antigen. The third method combined the first two methods using GAR/Au20 as th second layer and RAP/Au20 as the third layer which increased the amount of bound gold and enhanced the red colour, providing a better picture. The use of gold-labelled antibodies in double immunostaining has great potential value for many studies including that of the diffuse neuroendocrine system of the gut.

Colloidal gold for the detection of proteins on blots and immunoblots.

Immunoblotting techniques, in which electrophoretically separated proteins are transferred to the surface of nitrocellulose or nylon membranes ("Western" blots), are becoming increasingly popular in the analysis of protein systems. Transfer techniques in protein blotting have been covered in this series ( Chapter 20 in vol. 1, and Chapters 28 and 29 in this volume). In this chapter we will describe the use of the colloidal gold marking system for the detection of proteins on blots.

Postural abnormalities and contraversive pushing following right hemisphere brain damage.

We investigated the presence of postural abnormalities in a consecutive sample of stroke patients, with either left or right brain damage, in relation to their perceived body position in space. The presence or absence of posture-related symptoms was judged by two trained therapists and subsequently analysed by hierarchical classes analysis (HICLAS). The subject classes resulting from the HICLAS model were further validated with respect to posture-related measurements, such as centre of gravity position and head position, as well as measurements related to the postural body scheme, such as the perception of postural and visual verticality. The results of the classification analysis clearly demonstrated a relation between the presence of right brain damage and abnormalities in body geometry. The HICLAS model revealed three classes of subjects: The first class contained almost all the patients without neglect and without any signs of contraversive pushing. They were mainly characterised by a normal body axis in any position. The second class were all neglect patients but predominantly without any contraversive pushing. The third class contained right brain damaged patients, all showing neglect and mostly exhibiting contraversive pushing. The patients in the third class showed a clear resistance to bringing the weight over to the ipsilesional side when the therapist attempted to make the subject achieve a vertical posture across the midline. The clear correspondence between abnormalities of the observed body geometry and the tilt of the subjective postural and visual vertical suggests that a patient's postural body geometry is characterised by leaning towards the side of space where he/she feels aligned with an altered postural body scheme. The presence of contraversive pushing after right brain damage points in to a spatial higher-order processing deficit underlying the higher frequency and severity of the axial postural abnormalities found after right brain lesions.

Contraversive pushing and inattention of the contralesional hemispace.

The aim of this study was to investigate, in 114 stroke patients, the frequency of occurrence of a largely unknown neurological disorder, characterized by a postural imbalance due to a 'pushing away' reaction of the body towards the contralesional side of space, in function of hemispheric lesion localization and gender. The study also investigate the relation of this contraversive pushing with active movement, somatosensory perception deficits and, in particular, inattention of contralesional hemispace and body. The similarity of the presence of contraversive pushing and the syndrome of spatial hemineglect together with a gender-related differentiation suggest the existence of a "pusher syndrome", in which the pathophysiology points in the direction of a spatial higher-order processing deficit, related to spatial inattention, underlying the higher frequency and severity of contraversive pushing after right brain lesions.

Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes.

A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

Lipid and lipoprotein changes after long-term weight reduction: the influence of gender and body fat distribution.

OBJECTIVES: This study was designed: 1) to evaluate the effect of weight loss on body fat distribution, 2) to determine whether indices of body fat distribution can be considered as a prognostic indicator for the ability to lose weight and 3) to evaluate whether a change of body fat distribution is associated with changes in plasma glucose, lipids and lipoproteins in both sexes in order to evaluate a gender difference. METHODS: 63 obese subjects (41 women and 22 men) were treated on an outpatient basis with an energy-reduced, protein-enriched low calorie diet (3150-4200 kJ/day) for a 6-month period. They were divided in different groups according to gender and body fat distribution using the waist-to-hip circumference ratio. RESULTS: Body fat topography can be altered by dieting, but not by more than it increases when a person gains weight. Body fat distribution seems to be a significant prognostic indicator for the ability to lose weight in women but not in men. Although body weight and the waist-to-hip circumference decreased significantly, no relationships were found between percent decrease in these parameters and percent changes in plasma glucose, lipids and lipoproteins. CONCLUSION: We conclude that an important caloric deficit may lead to a series of metabolic improvements but that gender and the type of fat distribution are important confounding factors in the prediction of metabolic success.

FerriDye: colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes.

A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD460 nm = 0.5, followed by Perls' reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell.

We describe a new approach to probe the molecular biology of the living cell that uses small colloidal gold particles coupled to specific ligands. They are visualized in cells by bright-field, video enhanced contrast microscopy. We describe the basic aspects of the technique and provide examples of applications to intracellular motility, cell membrane dynamics, receptor translocation, internalization, and intracellular routing. We also provide examples of the use of this approach in immunospecific labelling of cells and tissue sections.