Hyperphagocytic haemocytes in Manduca sexta.

We have discovered a new type of haemocyte in the larval stage of the tobacco hornworm moth Manduca sexta that has extreme phagocytic ability; each cell can engulf up to 500 bacteria. This level of phagocytosis may be unprecedented among animal cells. Although these hyperphagocytic cells (HP) only represent about 1% of the circulating haemocytes, they are responsible for sequestering the majority of the bacteria by circulating haemocytes when non-pathogenic, heat-killed Escherichia coli are injected into the haemolymph. Extreme phagocytosis by HP is not limited to Gram-negative bacteria since heat-killed Staphylococcus aureus as well as positively and negatively charged microspheres are also highly phagocytosed. Evidence is presented to show that phagocytosis by HP is involved in the early stages of nodule formation in infected insects. In addition, HP are also present in non-infected insects, characterised by their distinctive spreading morphology, which becomes impaired following hyperphagocytosis of bacteria. This is the first time that a dedicated "professional" phagocytic class of haemocyte has been reported for an invertebrate. The importance of these specialised cell types in the M. sexta immune response and their role in nodule formation is discussed.

Chemical characterisation of sodium starch glycolate particles.

The internal and surface chemical compositions of three sodium starch glycolate (SSG) products, Explotab, Primojel and Vivastar P were studied using scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), time of flight secondary ion mass spectrometry (TOF-SIMS) and 23Na nuclear magnetic resonance spectroscopy (NMR). The surfaces of Explotab and Primojel contained very distinct features containing Na and Cl, however, Primojel also contained features which contained Na which may reflect the presence of Na glycolate and/or Na citrates. Vivastar P contained relatively few surface Cl containing features. Analysis of cross-sections of the particles showed that Na appeared to be uniformly distributed throughout the particles of all the products. Additionally, there was a significant concentration of Cl in the periphery of Explotab and Primojel. In the case of Vivastar P, significant levels of Na and Cl were detected in the internal regions of the particles which, together with 23Na NMR, suggests that NaCl is uniformly distributed within Vivastar P. 23Na NMR also suggested that the ratio of organic Na to NaCl was considerably lower in Vivastar P than Primojel and Explotab. Overall, even though all these three products satisfy the pharmacopeial descriptions of SSG, these studies suggest that Primojel and Explotab exhibit different chemical compositions to Vivastar P. Since the three products studied are reported to be prepared from potato starch, the apparent differences in chemical composition probably reflect the different manufacturing processes used, however, batch to batch variations may account for some of the subtle differences.

Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.

Previous attempts to express the toxin complex genes of Photorhabdus luminescens W14 in Escherichia coli have failed to reconstitute their oral toxicity to the model insect Manduca sexta. Here we show that the combination of three genes, tcdA, tcdB, and tccC, is essential for oral toxicity to M. sexta when expression in E. coli is used. Further, when transcription from native toxin complex gene promoters is used, maximal toxicity in E. coli cultures is associated with the addition of mitomycin C to the growth medium. In contrast, the expression of tcdAB (or the homologous tcaABC operon) with no recombinant tccC homolog in a different P. luminescens strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer that P. luminescens K122 carries a functional tccC-like homolog within its own genome, a hypothesis supported by Southern analysis. Recombinant toxins from both P. luminescens K122 and E. coli were purified as high-molecular-weight particulate preparations. Transmission electron micrograph (TEM) images of these particulate preparations showed that the expression of tcdAB (either with or without tccC) in E. coli produces visible approximately 25-nm-long complexes with a head and tail-like substructure. These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin itself or is an activator of the complex. The implications for the potential mode of action of the toxin complex genes are discussed.

An unusual helical micro-organism found in the gut lumen of human subjects.

An earlier report described the discovery of a micro-organism in the form of a double helix in human small bowel biopsies. Mucosal biopsies of the stomach and small bowel obtained from patients with rheumatic diseases and dyspepsia by enteroscopy and gastroscopy were fixed for scanning electron microscopy to investigate the organism further. In 62% of biopsies, an organism in the form of a double helix with bifid ends, 5-30 microm long, was found lying free on the surface of the mucosa. The organism has been demonstrated in the stomach, duodenum and small bowel. Flagella were never seen to be associated with the organism. In spite of its helical form, the organism lacks many of the factors associated with spirochaete morphology. It is suggested that this, as yet unnamed organism, may be found throughout the length of the digestive tract. Its pathological significance is not known.

The incorporation of a stent-graft into the porcine aorta and the inflammatory response to the endoprosthesis.

The fate of a non-sutured anastomosis and the inflammatory response to an endoprosthesis in a porcine aorta was determined. Self-expanding aorto-aortic endovascular prostheses were deployed into the infrarenal aorta of Large White pigs by the transfemoral route, in accordance with Home Office regulations. Animals were followed up at intervals to 1 year. The aortas were explanted and specimens were subjected to light microscopy. Thirteen animals, mean weight 103 kg, underwent placement of a prosthesis. There was early evidence of a neointima at 4 weeks and a well-developed neointima by 12 weeks, with complete incorporation of the endoprosthesis. The early acute inflammatory reaction seen at 4 weeks had become chronic by 12 and was largely absent by 26 weeks. There was no evidence of atypia. Complete incorporation of the non-sutured anastomosis is seen in this model. The acute inflammatory reaction to the prosthesis seen at 4 weeks had largely subsided by 26 weeks.

On the role of branched-chain amino acids in protein turnover of skeletal muscle. Studies in vivo with L-norleucine.

The effect of L-norleucine, an isomer of leucine, on protein metabolism in vivo was studied in suckling rats. Rats were injected subcutaneously with various doses of L-norleucine (0.5 and 5.0 mumol/g body wt.) every 12 h from 3 to 15 days post partum. Protein concentration, amino acid concentrations, and incorporation of [3H]tyrosine into protein were analyzed in liver, muscles of thigh and small intestine. Amino acid concentrations and insulin levels in serum were also measured. At 5 days of age, norleucine induced an increase in protein concentration of skeletal muscle with an increased incorporation of [3H]tyrosine into protein indicating an accelerated protein synthesis. Changes in protein metabolism were paralleled by alterations in the amino acid pattern of this tissue. When protein concentration and protein synthesis were increased in skeletal muscle, protein concentration of small intestine was decreased, accompanied by elevated levels of amino acids in tissue. Protein synthesis of small intestine was not altered by the norleucine treatment. The results suggest a close interrelationship between skeletal muscle and small intestine with respect to protein turnover. The effects of norleucine were less pronounced at 10 and 15 days of age, which indicates a metabolic adaptation to the treatment. Alterations in amino acid concentrations of tissue due to changes in protein metabolism were not uniform but tissue-specific. Current concepts for explaining the effects of branched-chain amino acids (BCAA) on protein turnover in skeletal muscle are based on the assumption that the BCAA or leucine alone might become rate-limiting for protein synthesis in muscle under catabolic conditions. The amino acid analogue norleucine, however, cannot replace any of the BCAA in protein. Additionally, norleucine affected protein metabolism in highly anabolic organisms. Therefore, the present thoughts on this issue appear to be incomplete.

Post-meiotic nucleo-cytoplasmic interaction in Cosmos bipinnatus : Early events at the nuclear envelope.

An interaction involving the nuclear envelope and spherical double-membrane bound inclusions takes place in the cytoplasm of post-meiotic male microspores of Cosmos (tribe Heliantheae, sub-tribe Coreopsidinae). The identity of the spherical inclusions has yet to be fully established, but they closely resemble profiles elsewhere in the cytoplasm, themselves presumably derived from the mitochondrial population of the premeiotic pollen mother cells. Both the cytoplasmic and nucleaar-associated inclusions regularly contain a central 'vesicle', formed by an ingagination of their bounding membranes. The interaction, which occurs immediately prior to the deposition of the primexine of the pollen wall, involves the adhesion of the inclusions to the nuclear surface. Experiments with osmotically disrupted cells reveal that the inclusions are firmly bound to the envelope and, at the points of contact, electron opaque granules are regularly present. Frequently elements of the chromatin may be observed in juxtapostion to these points of contact, but on the inner face of the envelope. The interaction in Cosmos is proposed to constitute part of the process by which the cytoplasm and its content are realigned to the new "gametophylic" style of growth.

An evaluation of Coomassie Brilliant Blue as a stain for quantitative microdensitometry of protein in section.

The specificity and stoichiometry of the binding of Coomassie Brilliant Blue (CBB) to protein in section has been examined using both frozen protein matrices and plant material. The maximum adsorbance of the stain, bound and in solution, was found to be 620 nm although variation in the results at this wavelength necessitated measurements to be made at 600 nm. After enzyme treatments of sectioned plant material embedded in resin, all CBB-binding biological material was shown to be sensitive to non-specific protease. The relationship between optical density at 600 nm and section thickness was tested statistically against the Lambert-Beer law, using microdensitometry of cryostat-sectioned, frozen genatine solution. The analyses showed conclusively that, under these conditions, CBB adheres strongly to the Lambert-Beer relationship. CBB may thus be considered as a very specific protein stain, eminently suited both to cytological observation and quantitative microdensitometry.

Post meiotic nucleo-cytoplasmic interaction in Pinus banksiana: The secretion of RNA by the nucleus.

Using digestion with specific enzymes, the chemical nature has been resolved of components of the nuclear invaginations formed in post-meiotic microspores of Pinus banksiana (Lamb.). The content of the invaginations is formed almost entirely of RNA, while the main bulk of the granular material investing the narrow part of the invaginations is also RNA. The membranes composing the narrow portion of the invaginations are themselves sensitive to RNase.These data are considered as supporting further the hypothesis that the invaginations are involved in the passage and organisation of information-carrying macromolecules.