Detection of mucosal and serum antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b by enzyme-linked immunosorbent assay

A sensitive enzyme-linked immunosorbent assay (ELISA) with rough-surfaced glass beads used as the solid phase was developed for detection of IgG, IgM, and IgA antibodies to Haemophilus influenzae type b capsular polysaccharide (HITB-CP). A successful method for indirect coating of glass bead surfaces with HITB-CP, and parameters affecting the specificity and sensitivity of the assay are described. This ELISA system proved to be 100 times as sensitive as the standard indirect fluorescent-antibody assay. The assay was applied to the measurement of antibodies to HITB-CP in serum and nasal secretions and proved to be a useful tool in the evaluation of immunological response to HITB infection.     

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2–4 μg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.     

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: Comparison of wild-type protein and active-site mutant D27E

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl₂ concentration-dependence of the ¹H–¹⁵N HSQC spectra of the wild-type DHFR–folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased K_m and K_d values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250 MPa together with negative activation volumes of ? 4.0 or ? 4.8 mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7 mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.     

Ribosomal acidic proteins of eucaryotic cells

Summary By the method of ethanol-salt extraction with ion-exchange chromatography on CM-cellulose an acidic protein of pea 80S ribosomes was isolated. This protein located in the large subunit, had a molecular weight of 14 000 and an IEP of 4.7. The protein is partially phosphorylated, alanine-rich and has methionine at the N-terminal position. Based on these characteristics and on the comparative study of tryptic hydrolyzates of the plant protein and E. coli L7/L12, the protein so obtained is found to be homologous to the L7/Ll2 of the procaryotic ribosomes.     

A pituitary-salivary mixed gland induced by tissue recombination of embryonic pituitary epithelium and embryonic submandibular gland mesenchyme in mice

Renal subcapsular syngrafts of Day 9 to 11 mouse embryonic pituitary epithelium with Day 14 mouse embryonic submandibular gland mesenchyme produced mixed organs that include residual cleft structure surrounded by anterior pituitary cells some which are stained by anti-ACTH antiserum and submandibular gland-like structure with differentiated acinar cells which are stained by anti-alpha-amylase antiserum. However, when Day 8.5 or 12 embryonic pituitary epithelium was recombined with submandibular gland mesenchyme and syngrafted, development of submandibular gland-like or anterior pituitary tissues resulted, respectively. Thus, during organogenesis of the mouse anterior pituitary, there exists a developmental stage (Day 8.5-11 in utero), when prospective pituitary epithelium can respond to heterotypic submandibular gland mesenchyme with the development of a submandibular gland-like tissue.     

The effect of prednisolone and metyrapone on FSH release induced by the administration of LRH.

The response of follicle-stimulating hormone (FSH) to a single injection of synthetic LRH was established in 7 and 6 women following an intramuscular dose of 0.2 mg and 0.1 mg. The secretion of FSH was greater in the group injected with 0.2 mg LRH than in the group injected with 0.1 mg. On the other hand, the response of FSH to a single injection of LRH (0.1 mg/subject) was established in 7 men before and after the pretreatment with metyrapone for one dat (4.5 g/subject). Pretreatment with metyrapone provoked a hypersecretion of FSH following a single injection of synthetic LRH. Seven women, 21--48 years of age who were treated with prednisolone for at least 1.5 months were examined for the responsiveness of the anterior pituitary to a single injection of synthetic LRH (0.2 MG). The secretion of FSH was not suppressed and the maximal serum level of FSH was observed 60 min after LRH injection.     

Onset and development of cannibalistic behaviour in early life stages of yellowtail

The onset and development of cannibalistic behaviour were observed in early life stages of yellowtail Seriola quinqueradiata . Cannibalistic behaviour was divided into four actions, i.e aim, chase, nip and ingestion. The frequency of chase was used as an index of cannibalistic behaviour because it always appeared in every sequence of cannibalism, although a sequence of cannibalistic behaviour sometimes stopped before nip or ingestion. No cannibalistic behaviour was observed during the larval phase until day 22 after hatching (when fish were 9.6mm T.L.) either in a rearing pond or in experimental tanks. The onset of cannibalistic behaviour was observed on day 23, coinciding with metamorphosis from the larval to juvenile phase, and it developed until day 39, with a tentative decrease between day 33 and day 36. This inverted peak corresponded roughly to the development of schooling behaviour after day 33, which was determined by distance to the nearest neighbour. In the rearing pond, suffocation of a cannibal by its prey, appeared from day 23. Field observation of juvenile yellowtails aggregating around floating seaweeds showed that cannibalism occurred in three out of 10 schools, in which six cannibals were found among 194 fish. Cannibalistic behaviour in early life stages of yellowtail may occur as a final phase of inter-individual interference and may have a role for size selection of a School member.