Tissue and subcellular immunolocalisation of enzymes of lignin synthesis in differentiating and wounded hypocotyl tissue of French bean (Phaseolus vulgaris L.)

Antisera raised againstl-phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and a cationic cell-wall peroxidase, which had all been purified from suspension-cultured cells of French bean, have been used to carry out immunogold localisations in the growing plant. Immunoglobulin-G fractions were prepared from each antiserum and used to study the distribution of the enzymes in differentiating and wounded hypocotyls by immunogold techniques and visualisation by both light and electron microscopy. Following silver enhancement to amplify the signal, proteins were detected by confocal microscopy in both developing (pre-xylem/ phloem) and later metaxylem stelar tissue.l-Phenylalanine ammonia-lyase and C4H also accumulated in cells adjacent to metaxylem, presumably involved in maintaining a supply of phenylpropanoid precursors to the enucleated xylem for further lignin synthesis. In these cells, PAL subunits were cytosolic although some were associated with endomembrane. Cinnamate-4-hydroxylase was wholly associated with membrane and particularly high concentrations were found in the Golgi bodies. The cationic peroxidase accumulated in xylem at sites of secondary thickening and in the middle lamella. The three proteins are also involved in defensive lignification. Thus when visualised by light microscopy, PAL and C4H were seen to accumulate to high levels throughout the cell types in wound sites and especially in the epidermal cells. An even more intense general distribution was found upon hyperinduction of wounded cells with-aminooxy--phenylpropionic acid. At the subcellular level, PAL was found to be localised in the cytosol in the wounded cells; however, because of the loss of membrane through mechanical damage, association with membrane structures, particularly endoplasmic reticulum, in unwounded cells is not entirely ruled out. Cinnamate-4-hydroxylase was associated with membranes when these were preserved. In wounded tissue, the peroxidase was found at the growing edges of tylose-like structures in the vascular xylem.Abbreviations AOPP -aminooxy--phenylpropionic acid - C4H cinnamic acid-4-hydroxylase - CHS chalcone synthase - GRP glycine-rich glycoprotein - HRGP hydroxyproline-rich glycoprotein - Ig immunoglobulin - PAL phenylalanine ammonia-lyase G.P.B. thanks the Agicultural and Food Research Council for support.     

Relationships between ligninolytic activities of Lentinula spp. and biotransformation of pentachlorophenol in sterile soil

Ligninolytic activities in strains of Lentinula edodes were related to pentachlorophenol biotransformation in sterile soil and activities in L. lepideus. Strains of L. edodes secreting laccase and manganese peroxidase activities also metabolized pentachlorophenol (PCP) significantly ( P < 0.05). Strains of L. lepideus showed neither enzymic activities nor xenobiotic breakdown. Lentinula edodes strains inhibited by PCP at 5 mg 1^-1 in agar, tolerated 200 mg kg^-1 in soil. Strain LE2 metabolized more PCP in nitrogen-sufficient than nitrogen-limited culture: the reverse was observed with Phanerochaete chrysosporium BKM 1767. Relationships between ligninolytic activities and pentachlorophenol breakdown in L. edodes indicated a suitability for soil bioremediation treatments.     

Polymorphic Müllerian mimicry in a group of African butterflies: a re-assessment of the relationship between Danaus chrysippus, Acraea encedon and Acraea encedana (Lepidoptera: Nymphalidae)

Müllerian mimicry, in which there is convergence in coloration between unrelated unpalatable species, should lead to uniformity in appearance, not polymorphism, and so the occurrence in tropical Africa of unrelated species of unpalatable butterflies with corresponding polymorphic colour forms suggested a problem of special evolutionary interest. Field work in Uganda and Sierra Leone in 1964 72 demonstrated a statistical association between the occurrence and relative frequencies of polymorphic forms in Danaus chrysippus (Danainae) and Acraea encedon (Acraeinae) which was deemed as confirmation of a Müllerian relationship between them. There were, however, certain anomalies which at the time remained unresolved. Later, in 1976, it was discovered that what had been called A. encedon is in reality two sibling species. A. encedon and a new one, named as A. encedana. The two differ in the structure of both male and female genitalia and in the coloration and the food-plants of the larvae. The recognition of the additional species has enabled a re-assessment of the polymorphie Müllerian association with D. chrysippus. It emerges that, although there is a close qualitative and quantitative reciprocal mimetic relationship between A. encedana and D. chrysippus, the relationship between A. encedon and D. chrysippus is much weaker, and in places non-exitent. The possible origin of the mimetic polymorphism is discussed in terms of hybridization of previously allopatric and monomorphic populations which have met as a consequence of recent expansions of geographical range in all three species resulting from forest clearance and the spread of savanna-like conditions in previously forested areas.     

Non-invasive detection of malignancy by identification of unusual CD44 gene activity in exfoliated cancer cells.

OBJECTIVE--To investigate non-invasive detection of cancer by testing for unusual CD44 gene activity in a clinical sample as an indicator of exfoliated tumour cells. DESIGN--Case-control study. SUBJECTS--44 unselected, consecutive patients with bladder cancer and 46 people with no evidence of neoplasia. MAIN OUTCOME MEASURE--Presence or absence of large CD44 gene products containing exon 6 derivatives in urine samples. RESULTS--Novel abnormalities in the pattern of expression of this gene, seen specifically in tumour tissue, led to cloning of a newly recognised coding region in it (exon 6). This was tested as a probe for detection of exfoliated malignant cells in naturally voided urine. CD44 gene products extracted from the urine and amplified with polymerase chain reaction contained predicted electrophoretic band of 735 base pairs in 40 of the 44 patients with bladder cancer (sensitivity 91%). Products from 38 of the 46 people with no evidence of neoplasia showed no such band (specificity 83%). CONCLUSIONS--Unusual activity of the CD44 locus in neoplasia and malignancy is confirmed, and techniques for the analysis of such activity can enable non-invasive investigation of patients for primary or recurrent bladder cancer or for other tumours that shed neoplastic cells into body fluids.     

Waiting times: monitoring the total postreferral wait.

OBJECTIVES--To determine whether the period spent on the true inpatient waiting list is a valid indication of the total time that patients have to wait for an operation; and to assess the feasibility of monitoring the total "postreferral waiting time" by using existing computerised information systems. SETTING--Three randomly selected Scottish hospitals. SUBJECTS--Waiting list patients admitted to hospital for operations during June to August 1993 in six major specialties, separate attention being focused on cataract operations and hip and knee replacements. MAIN OUTCOME MEASURE--The total time that patients have to wait for an operation after the initial general practitioner referral--the postreferral waiting time--compared with that spent at the final stage of the process on the true inpatient waiting list. RESULTS--In the specialties investigated roughly half (58 days; 53%) of the average postreferral wait of 110 days was spent on the true inpatient waiting list, one third (35 days; 32%) being spent on the outpatient waiting list and one sixth (17 days; 15%) waiting between waiting lists. Only a quarter of cataract patients (73/292) were treated within three months of general practitioner referral compared with over three quarters (228/292) within three months of being placed on the inpatient waiting list. Nevertheless, within a year over 99% of patients (290) had been treated whichever date was taken as the starting point. CONCLUSIONS--Monitoring postreferral waiting times would provide a much more accurate picture for purchasers and patients of waiting times for treatment than is obtained by focusing exclusively on the true inpatient waiting list and facilitate fairer comparisons between NHS trusts in national league tables. Stringent national and local monitoring is essential to ensure (a) that future reductions in the time waiting on true inpatient waiting lists are not gained at the expense of longer periods waiting to be placed on the lists, and (b) that no increases occur in the number of patients placed instead on deferred waiting lists or exempted from the normal maximum waiting time guarantees.     

Higher professional training in general practice: provision of master's degree courses in the United Kingdom in 1993.

Review of a 1993 survey of the 29 United Kingdom departments of general practice (or equivalent) identified seven master''s degree courses available for general practitioners. Up to another 11 are planned within the next five years. Around 50 general practitioners undertake all such courses at any one time. Possible reasons for this low uptake include cost, lack of flexibility of courses, and the prospect of writing a thesis. Appropriate master''s courses are essential to the future development of general practice, and this paper postulates the characteristics of an "ideal" course.     

Familial unbalanced translocation t(8;15)(p23.3;q11) with uniparental disomy in Angelman syndrome

A 29-year-old male with Angelman syndrome and an unbalanced reciprocal translocation, 45,XY,-8,-15,+der(8),t(8;15)(p23.3;q11)pat, was evaluated with DNA studies. These showed the underlying mechanism to be paternal uniparental disomy. This is the second case reported of Angelman syndrome that has resulted from a familial unbalanced reciprocal translocation.     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

1.56 Å structure of mature truncated human fibroblast collagenase

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 Å resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded β-sheet and three α-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase. © 1994 Wiley-Liss, Inc.