Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat-shocked xenopus laevis embryos

In the present study we have characterized the synthesis of members of the HSP30 family during Xenopus laevis development using a polyclonal antipeptide antibody derived from the carboxyl end of HSP30C. Two-dimensional PAGE/immunoblot analysis was unable to detect any heat-inducible small HSPs in cleavage, blastula, gastrula, or neurula stage embryos. However, heat-inducible accumulation of a single protein was first detectable in early tailbud embryos with an additional 5 HSPs at the late tailbud stage and a total of 13 small HSPs at the early tadpole stage. In the Xenopus A6 kidney epithelial cell line, a total of eight heat-inducible small HSPs were detected by this antibody. Comparison of the pattern of protein synthesis in embryos and somatic cells revealed a number of common and unique heat inducible proteins in Xenopus embryos and cultured kidney epithelial cells. To specifically identify the protein product of the HSP30C gene, we made a chimeric gene construct with the Xenopus HSP30C coding sequence under the control of a constitutive promoter. This construct was microinjected into fertilized eggs and resulted in the premature and constitutive synthesis of the HSP30C protein in gastrula stage embryos. Through a series of mixing experiments, we were able to specifically identify the protein encoded by the HSP30C gene in embryos and somatic cells and to conclude that HSP30C synthesis was first heat-inducible at the early tailbud stage of development. The differential pattern of heat-inducible accumulation of members of the HSP30 family during Xenopus development suggests that these proteins may have distinct functions at specific embryonic stages during a stress response.     

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.     

Mycorrhizal associations in Hong Kong Fagaceae

Polyphenols histochemically detected in fresh uninfected roots of Quercus, Castanopsis and Lithocarpus growing in Hong Kong and shown to be condensed tannins were found mainly as intracellular material in the cells of the root cap, the epidermal layer and the endodermis. The cell walls of the outer cortex and the endodermis also contained suberin. Following invasion by compatible ectomycorrhizal symbionts, condensed tannins disappeared from cells of the root cap and the epidermal layer but hyphae were prevented from colonizing the cortex presumably due to suberin barriers. In vitro experiments indicated that a number of broad-host ectomycorrhizal fungi could utilise various polyphenolic compounds, including tannins found in the root exudates of the host trees, with different degrees of efficiency.     

Nutrients and heavy metal contamination of plants and sediments in Futian mangrove forest

An ecological survey was carried out to determine the levels of nutrients and heavy metals in the sediments and leaf tissues of two dominant mangrove plant species, Kandelia candel and Aegiceras corniculatum, in Futian mangrove forest, Shenzhen, the People's Republic of China. The spatial and seasonal variations of these elements were also investigated. The results show that there was no major difference between two sampling sites 150 m apart. In both sites, the sediment concentrations of total and NH₄ ⁺-N, total and extractable P, total and extractable K, total organic carbon were consistently higher in the landward locations and decreased gradually towards the sea. The sediment sample collected at the seaward edge of the mangrove plant community had the lowest levels of nutrient and organic matter. The vertical variations (from the land to the sea) of sediment heavy metals were less obvious and no particular trend could be identified. Extremely high contents of Cu, Cd, Pb, Cr and Zn were found at certain locations, suggesting the occurrence of some local contamination. The mean total metal concentrations in sediments decreased in the order Mn > Zn > Cu > Cr = Pb > Cd for the sample locations. Most of the heavy metals were not in a bioavailable form as the concentrations of extractable metals were relatively low (< 1% of total metals). Pb, Cr and Cd were not detected in leaf samples. Leaf C, N, P and K contents were similar between the two species and no significant difference was found among locations, although A. corniculatum seemed to have lower Mn concentrations than K. candel. With reference to temporal variations, no significant difference in sediment concentrations of some nutrients and metals was found between the spring and autumn seasons.     

Comparison of the effect of calcium(II) and manganese(II) ions on trypsin autolysis

The effect of Mn²⁺ and Ca²⁺ ions on the rate of trypsin autolysis was studied at pH 7.0 and at 34.4-60.2°C. For comparison, the kinetic constants of esterolytic activity of trypsin in the presence of the metal ion were determined at pH 7.4 and at 36° and 40°C. There was no significant difference in the rate of autolysis between Mn²⁺ and Ca²⁺ in the temperature range 34-47°C, but at 56.8° and 60.2° autolysis was slightly more rapid in the presence of Mn²⁺. The Mn²⁺ or Ca²⁺ ion bound to trypsin is supposed to control the conformation and thereby the stability and the activity of the enzyme. This indirect effect of Mn²⁺ and Ca²⁺ is discussed on a structural basis of the enzyme molecule.     

Oxygen-binding and immunological properties of complexes between dextran and animal haemoglobins.

Complexes of dextran 20 000 with haemoglobins of sheep, rabbit, dog, bovine and human origin were prepared through alkylation of haemoglobin by N-bromoacetylaminoethylamino-dextran. The yields were uniformly high. Complex-formation in each case was accompanied by the disappearance of reactive thiol groups on the haemoglobin, and by an increase in the affinity of the haemoglobin for oxygen. The immunological properties of dog, rabbit and sheep dextran-haemoglobin were investigated in both homologous and heterologous species. The complexes were found to be non-immunogenic in the homologous species. In heterologous species the anti-haemoglobin response induced by each complex was generally of a similar level to that induced by the haemoglobin alone.     

A single mutation affects L-serine deaminase, L-leucyl-, L-phenylalanyl-tRNA protein transferase, and proline oxidase activity in Escherichia coli K-12.

A mutation at a single locus, wyb, results in several phenotypic changes in Escherichia coli K-12. The Wyb- phenotype includes: (i) an increase in L-serine deaminase activity, together with a loss of inducibility by L-leucine; (ii) an absence of L-leucyl-, L-phenylalanyl-tRNA protein transferase activity; (iii) inducibility of proline oxidase by proline; and (iv) a loss of ability to use maltose as a carbon and energy source.     

Evaluation of human monoclonal antibody (2-139-1) in cutaneous melanocytic neoplasms in fixed tissue sections.

Despite the growing list of xenogeneic monoclonal antibodies (MAb) that recognize malignant melanoma-associated antigens (MAA) in formalin-fixed, paraffin-embedded tissue, none has been able to detect epitopes found in malignant melanomas and not in melanocytic nevi. A human MAb, 2-139-1, that showed promise in this regard was evaluated against 85 melanocytic neoplasms, including malignant melanoma and histological simulators, particularly Spitz's nevus. MAb 2-139-1 stained 18 (53%) of 34 melanomas, eight (57%) of 14 dysplastic nevi, six (38%) of 16 Spitz's nevi, and three (14%) of 21 banal nevi, which included three small congenital nevi. We observed a significant increasing trend in reactivity (% positive cells x intensity) associated with the potential for malignancy (p for linear trend = 0.002). We conclude that human MAb 2-139-1 is applicable to the study of melanocytic neoplasms in routinely processed tissue. Although the ability of this MAb to separate benign from malignant cells is not absolute, our results suggest that the expression of the 2-139-1 epitope may be an early event in melanocytic tumor progression.     

Location of the origin of replication for the 7.5-kb Chlamydia trachomatis plasmid.

The hypothetical origin of replication for the 7.5-kb plasmid common to Chlamydia trachomatis is believed to be in a region of the plasmid that contains four 22-bp tandem repeats preceded by an A-T-rich region. To test this hypothesis, replication of plasmid DNA in metabolically active reticulate bodies of the Lymphogranuloma venereum biovar of C. trachomatis was examined by electron microscopy. The results presented show that the origin of replication appears to be near the tandem repeats of pCHL2. In addition, replication of the 7.5-kb plasmid is unidirectional, and the copy number during replication is 7-10. The evidence presented suggests that C. trachomatis has a homologue to the Escherichia coli dnaA gene and that this homologue might be involved in replication of the C. trachomatis 7.5-kb plasmid.