Electrical consequences of spine dimensions in a model of a cortical spiny stellate cell completely reconstructed from serial thin sections

We built a passive compartmental model of a cortical spiny stellate cell from the barrel cortex of the mouse that had been reconstructed in its entirety from electron microscopic analysis of serial thin sections (White and Rock, 1980). Morphological data included dimensions of soma and all five dendrites, neck lengths and head diameters of all 380 spines (a uniform neck diameter of 0.1 m was assumed), locations of all symmetrical and asymmetrical (axo-spinous) synapses, and locations of all 43 thalamocortical (TC) synapses (as identified from the consequences of a prior thalamic lesion). In the model, unitary excitatory synaptic inputs had a peak conductance change of 0.5 nS at 0.2 msec; conclusions were robust over a wide range of assumed passive-membrane parameters. When recorded at the soma, all unitary EPSPs, which were initiated at the spine heads, were relatively iso-efficient; each produced about 1 mV somatic depolarization regardless of spine location or geometry. However, in the spine heads there was a twentyfold variation in EPSP amplitudes, largely reflecting the variation in spine neck lengths. Synchronous activation of the TC synapses produced a somatic depolarization probably sufficient to fire the neuron; doubling or halving the TC spine neck diameters had only minimal effect on the amplitude of the composite TC-EPSP. As have others, we also conclude that from a somato-centric viewpoint, changes in spine geometry would have relatively little direct influence on amplitudes of EPSPs recorded at the soma, especially for a distributed, synchronously activated input such as the TC pathway. However, consideration of the detailed morphology of an entire neuron indicates that, from a dendro-centric point of view, changes in spine dimension can have a very significant electrical impact on local processing near the sites of input.     

Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.     

Structure and identification of root bark of Quercus robur L.

The root bark structure of Quercus robur L. was analysed at different stages of root development and compared to the structure of stem bark. Root bark thickness varied considerably between different roots. Sclereid quantity decreased with increasing distance from the stem, which means it increased with age. Visible growth increments diminished with increasing distance from the stem. In lateral roots crystal quantity decreased with increasing distance from the stem. In lateral roots secondary phloem fibre length, sieve tube member length, and sieve tube diameter showed no regular trend. There were only a few basic structural differences between root and stem bark. The zone of cell differentiation (cell expansion, lignification) was wider in root bark; sieve tube collapse was delayed. In lateral root bark fewer sclereids were formed. The first-formed periderm often originated from deeper cell layers. Thus, primary elements were lacking after periderm formation. In root bark the phellem cell walls were of equal thickness. Thus, phellem lacked visible growth increments. Root bark phellem cells were slightly larger. The root phelloderm was more distinct. The secondary phloem fibres were slightly shorter than those in stem bark. Sieve tube members of stem and root bark were of similar length and diameter. The qualitative bark anatomical characters of oak root bark are suitable for root identifications. Due to minor structural differences between root and stem bark the characters must be used with care.     

Hydrogen fluoride effects on plasma membrane composition,ATPase activity and cell structure in needles of eastern white pine (Pinus strobus) seedlings

Eastern white pine (Pinus strobus L.) seedlings were grown in controlled environment growth cabinets and fumigated with 0.4 and 1.6 g m^–3 hydrogen fluoride for 2–28 days. Plasma membranes were isolated from needles of treated and control seedlings and their chemical composition and ATPase activity examined to determine early effects of hydrogen fluoride action. In plants treated for 2 days with both fluoride levels, ratios of plasma membrane free sterols:phospholipids and sterols:proteins were drastically higher than ratios in control plants. Seedlings treated with hydrogen fluoride for 8 days contained plasma membranes with elevated phospholipid:protein and sterol:protein ratios and their plasma membrane ATPase activity was higher than that of control plants. Prolonged, 28-day hydrogen fluoride treatment with 1.6 g m^–3 level was the only treatment which produced a drastic inhibition of plasma membrane ATPase activity. During the initial stages of hydrogen fluoride treatment, treated cells did not show alterations of ultrastructure which were previously shown in cells of plants treated with soil applied sodium fluoride. The results of the present study indicate that the plasma membranes may be among the initial sites of hydrogen fluoride injury to plants as well as initial sites of defense reaction.     

Modifications of S100-protein immunoreactivity in rat brain induced by tissue preparation

Immunocytochemistry using antibodies against various molecular forms of the Ca⁺⁺ and Zn⁺⁺-binding S100 proteins predominantly labelled astrocytes. However, especially in the neocortex the staining pattern is variable. Methods of tissue preparation have been evaluated with the aim to preserve as much S 100 immunoreactivity as possible. Optimal results were obtained after perfusion fixation with 4–5% aldehydes, 0.1M sodium cacodylate, 0.1% CaCl₂, pH 7.3. In such preparations, astrocytes were completely labelled including their lamellar compartments in large parts of the central nervous system. Ca⁺⁺-withdrawal had adverse affects on S100 immunoreactivity. Cryostat sections treated with EDTA-containing solutions before fixation showed that Ca⁺⁺-free S100 can apparently not be fixed to the tissue. Perfusion fixatives containing EDTA resulted in inhomogeneous loss of S100 staining, indicating a differential susceptibility of astrocytic subpopulations. A different type of reduction in S100 immunoreactivity occurred around large neocortical blood vessels. Perivascular defects in immunostaining occasionally appeared even after optimal fixation, but could be regularly provoked by mildly acidic fixation (pH 6.6) or prolonged barbiturate anaesthesia. These defects might be based on S100 release into the cerebrospinal fluid. Presumably under none of the conditions studied can the immunoreactivity of all S100-forms and-fractions be completely preserved in the tissue. However, recommendations are presented for optimizing tissue preparation, to the extent that premortal modifications affecting the stainability of astrocytes may be detected by S100 immunohistochemistry in fixed brain tissue.     

Role of the conserved C-repeat region of the M protein of Streptococcus pyogenes

The surface-located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C-repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M-protein-mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C-repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C-repeat domain, a region of the M-protein molecule distinct from the C-repeat domain confers upon S. pyogenes its ability to resist phagocytosis.     

Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose

Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N -linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O -linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1-4-linked digalactose moiety covalently linked to a 2,4-diacetamido-2,4,6-trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase ( galE ) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper-adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.     

Opc- and pilus-dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides

The interplay between four surface-expressed virulence factors of Neisseria meningitidis (pili, Opc, capsule and lipopolysaccharide (LPS)) in host cell adhesion and invasion was examined using derivatives of a serogroup B strain, MC58, created by mutation (capsule, Opc) and selection of variants. To examine the role of Opc and of additional expression of pili, bacteria lacking the expression of Opa proteins were used. The effects of different LPS structures were examined in variants expressing either sialylated (L3 immunotype) or truncated non-sialylated (L8 immuno-type) LPS. Studies showed that (i) pili were essential for meningococcal interactions with host cells in both capsulate and acapsulate bacteria with the sialylated L3 LPS immunotype, (ii) the Opc-mediated invasion of host cells by piliated and non-piliated bacteria was observed only in acapsulate organisms with L8 LPS immunotype, and (iii) expression of pili in Opc-expressing bacteria resulted in increased invasion. Investigations on the mechanisms of cellular invasion indicated that the Opc-mediated invasion was dependent on the presence of serum in the incubation medium and was mediated by serum proteins with arginine-glycine-aspartic acid (RGD) sequence. Cellular invasion in piliated Opc⁺ phenotype also required bridging molecules containing the RGD recognition sequence and appeared to involve the integrin αvβ3 as a target receptor on endothelial cells. These studies extend the previous observations on variants of a serogroup A strain (C751) and show that Opc mediates cellular invasion in distinct meningococcal strains and provide confirmation of its mechanism of action. This is the first investigation that evaluates, using derivatives of a single strain, the interplay between four meningococcal surface virulence factors in host cell invasion.     

An improved method for cell and spore counts during aerobic cultivation of crystal toxin producingBacillus sphaericus

Summary A simple improved method including sonication treatment is proposed to determine accurate cell and spore counts forBacillus sphaericus, a microorganism which can form cell aggregates. Sonication (10 watt power output) for 40 seconds after dilution of culture broth was effective in dispersing clumps of cells and spores without disruption. This improved method gave approximately 2 times higher cell and spore counts compared with the conventional counting methods (without sonication).