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Conjugation of folate to proteins permits receptor-mediated endocytosis via the folate receptor (FR) and delivery of the conjugate into the cytoplasm of cells. Since many cancers up-regulate the FR it has enabled the targeting of toxins to tumor cells resulting in specific cell death. However, current conjugation methods rely on chemistries that can affect certain catalytic subunits, such as the A-chain of the plant toxin gelonin. As a result many folate-targeted toxins are a compromise between receptor/ligand interaction and toxin activity. We describe the first example of folate conjugated to a protein via carbohydrate residues, using a novel SH-folate intermediate. The folate-gelonin conjugate retains over 99% of toxin activity in a cell-free translational assay compared with unmodified gelonin and is able to bind the FR at the same affinity as free folic acid (10(-10) m). Additionally, the conjugate exhibits prolonged inhibition of protein synthesis in FR positive cell lines in vitro. Folate linked to gelonin via amino conjugation exhibits the same affinity for FR as free folic acid but the toxin is 225-fold less active in a cell-free translational assay. The effect of different conjugation methods on toxin activity and the implications for folate targeting of other glycoproteins are discussed.

AbstractAt least 13 marine terraces or their remnants, with maximum shoreline altitudes of c.3 m, 60, 90, 140, 230, 320, 370, 460, 580, 660, 780, 870 and 1040 m, are preserved in the Waitutu district of southeast Fiordland. Alluvial terraces related to glaciation of the immediate hinterland cross the marine terraces and allow the terraces to be confidently matched to the glacial/interglacial sequence. Three well-preserved marine terraces of the Last Interglacial separate fluvioglacial terraces of the Last and Penultimate Glaciations. An uplift rate of 1.1 mm/yr, derived from the altitude of the fourth youngest terrace inferred to represent oxygen isotope stage 5e, is used to estimate the ages of the older marine terraces by linear extrapolation, up to > 900,000 years.The Waitutu marine terraces traced into southwestern Fiordland decrease markedly in height, to levels corresponding to an uplift rate of 0.6 mm/yr. The higher peaks of Fiordland conform to a summit-level surface which is interpreted to be, in effect, a marine terrace of very early Pleistocene age. The surface is broadly domed in a pattern matching the marine terraces of the south coast.Many of the strong structural features in the vicinity of southern Fiordland, associated with the convergent boundary between the Indo-Australian and Pacific plates, are of late Miocene age and belie the simple deformation pattern of essentially epeirogenic uplift the area has undergone in the Quaternary.

Two groups of patients with Dupuytren’s disease were treated by limited fasciectomy. A tourniquet was used for one group, and an elevated hand table without tourniquet for the other. Taking into account other factors controlling oedema it was discovered over a post-operative period of twenty-eight days that “Tourniquet” hands remained significantly increased in volume compared to “Non-Tourniquet” hands. Other facts of clinical interest also emerge from the trial and are discussed. It is concluded that the use of the elevated hand table without tourniquet, not only allows an unlimited and uninterrupted period for hand surgery, but also reduces the subsequent oedema.

Monoclonal antibodies are able to provide information on the surface topography of biopolymers. Antibodies capable of inhibiting the activities of cutinase and parathion hydrolase have been identified in order to probe the sites of pesticide inhibition. Kinetic studies were employed to determine whether organophosphate pesticide binding was directly prevented by antibody binding. Prior binding of chlorfenvinphos and methyl paraoxon to cutinase inhibited subsequent binding by the monoclonal anti-cutinase antibodies. The role of antibodies in probing enzyme structure is discussed.

The application to soft drinks of a microtitration plate enzyme-linked immunosorbent assay for the determination of quinine is described. Dilution of sample allowed direct and simple analysis. Intra- and inter-assay coefficients of variation were 5.7% and 6.7% respectively. The advantages and disadvantages of the immunological approach over conventional techniques are discussed.

Three antibody-conjugated enzymes (urease, alkaline phosphatase and horse radish peroxidase) were compared for their suitability in simple visual assessment of microtitration plate ELISA colours. The model system used was the ELISA determination of total glycoalkaloids in potato tubers. Probit analysis of results showed that peroxidase, with 2, 2-azinobis (3-ethylbenzthiazoline) 3-sulphonic acid (ABTS) as substrate, was the most useful of the three enzymes in terms of recognizing positive samples. Visual assessment was able to identify accurately all potato samples containing glycoalkaloids at greater than the chosen critical level. Visual assessment of ELISA results is applicable to many other areas of food analysis.

As a result of a previous trial in which factors influencing swelling of the hand after fasciectomy were studied it was found that unacceptable oedema occurred in hands elevated in a roller towel by night and a high sling by day. An alternative system of elevation in a vacuum splint was investigated and found to reduce post-operative oedema. The advantages and disadvantages of the vacuum splint in the management of the hand after surgery or injury are discussed.

The reproducibility of a commercially available kit utilising enzyme-linked immunosorbent assay (ELISA) for determination of total aflatoxins has been analysed. Peanut butter samples contaminated with aflatoxin at three concentrations (mean 5.8, 11.7 and 24.8 ng/g) were analysed on different occasions using four kits. Coefficients of variation at all concentrations ranged from 2.6% to 13.6% within replicate, 0.3% to 16.2% within plate and 6.7% to 10.2% between kits. Standard curve precision profiles showed a minimum coefficient variation (CV) of 2-4% and a working range (below 10% CV) covering almost the entire standard curve.

Two suitably protected building blocks (11and 33) for the preparation of amide‐linked heptaglucoside mimetic 2, an analogue of the naturally occurring phytoalexin elicitor 1a, were readily accessible by glycal chemistry. Sequential elongation of terminal glucuronide 21with laminaribiosyl hemiaminal 33and anomeric amine 11by EDC/HOBt‐catalyzed condensation and two‐step conversion of the C6‐OTr moiety into the corresponding carboxylate function afforded homogeneous carbopeptoid 2in high overall yield. It was found that replacement of the acetal linkages by the more rigid amide bonds destroys the phytoalexin‐elicitor activity.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a homophilic adhesion receptor that mediates leukocyte/endothelial cell interactions that take place during transendothelial migration. Recent reports have shown that the binding of certain anti-PECAM-1 antibodies results in up-regulation of integrin function on the surface of leukocytes and platelets, suggesting that PECAM-1 may be capable of transmitting information into the cell following its engagement. PECAM-1 isolated from resting or activated but nonaggregated platelets was phosphorylated predominantly on serine residues; however, PECAM-1 derived from activated, aggregated platelets was strongly phosphorylated on tyrosine. Synthetic tyrosine-phosphorylated peptides derived from five different regions within the cytoplasmic domain of PECAM-1 were screened for their ability to associate with cytoplasmic signaling molecules. The protein-tyrosine phosphatase SHP-2 was found to interact specifically with two different PECAM-1 phosphopeptides containing highly conserved phosphatase-binding motifs on PECAM-1 with the sequences VQpY663TEV and TVpY686SEV. More important, SHP-2 bound not only PECAM-1 phosphopeptides, but also became associated with full-length cellular PECAM-1 during the platelet aggregation process, and this interaction was mediated by the amino-terminal Src homology 2 domains of the phosphatase. Since SHP-2 normally serves as a positive regulator of signal transduction, its association with activated PECAM-1 suggests a number of potential mechanisms by which PECAM-1 engagement might be coupled to integrin activation in vascular cells.

Arthrobacter crystallopoietesgrowing exponentially as cocci were changed to rods by adding succinate to the medium. Cells were sampled before, during, and after this transition for Gram-staining and ultrastructural studies. Cells were Gram stained by the standardized method of Bartholomew, and all samples were fixed and prepared for thin sectioning in an identical manner. Cocci were gram positive, and thin sections demonstrated a gram-positive type of cell wall having an average thickness of 31 nm. Cells sampled during morphogenesis appeared as cocci with most having a single rodlike projection. The coccus portion of these transition cells was gram positive and bound by a gram-positive type of wall having an average thickness of 29 nm. The rodlike projection of the transition cells appeared to be gram negative; it was also surrounded by a gram-positive type of wall, but its average thickness was only 22 nm. Gram-negative rods of the type species, Arthrobacter globiformis, were also examined and found to produce a gram-positive type of wall with a 19-nm average thickness. Evidence for the trilaminar region, characteristic of most gram-negative bacterial cell walls, was totally lacking in both species. These results suggest that variations in cell wall thickness may be an important contributing factor to the variable Gram-staining characteristics of this genus.

The notes of 158 patients who had undergone surgery of the parotid gland for benign and malignant disease were studied to discover the circumstances in which the facial nerve might be injured.Limited surgery carried as great a risk of injury to the facial nerve as during extended surgery. Extended surgery provided greater risk of injury to the facial nerve on secondary exploration (71 per cent) than on primary exploration (6.5 per cent).It is concluded that every primary exploratory procedure of the parotid gland should be a formal conservative parotidectomy unless the tumour is obviously malignant.

It has been recognised for a long time that damage to the median or ulnar nerves is usually followed by slowing of growth of the fingernails. In the past, this has always been ascribed to immobilization of the appropriate digits, rather than to a neurotrophic factor. No other nail changes appear to have been described in association with nerve damage. We present two patients with marked nail changes following median nerve injury, in which immobilization does not appear to be responsible.