Immunomodulation in patients receiving intravenous Bryostatin 1 in a phase I clinical study: comparison with effects of Bryostatin 1 on lymphocyte function in vitro

Summary Bryostatin 1 is a protein kinase C activator that inhibits growth of tumour cells and activates lymphocytes in vitro, properties that have encouraged its use in phase 1 clinical studies as an anticancer agent. We investigated interleukin-2(IL-2)-induced proliferation and lymphokine-activated killer (LAK) cell activity in peripheral blood mononuclear cells (PBMC) from cancer patients receiving Bryostatin intravenously. After Bryostatin administration both LAK generation and proliferation were enhanced when patients' PBMC were stimulated with IL-2 in vitro. However, when normal donors' PBMC were cultured in vitro in the presence Bryostatin and IL-2, LAK induction was inhibited while IL-2-driven proliferation was increased. These effects were also seen following only 2 h exposure to Bryostatin and could be elicited by conditioned medium from Bryostatin-pretreated cells. Neither IL-4 nor interferon was detected in the conditioned medium. Bryostatin in vitro was found to increase expression of IL-2 receptors on CD4⁺, CD8⁺ and CD56⁺ cells and augment the proportion of CD8⁺ cells in conjunction with IL-2. We conclude that Bryostatin in combination with IL-2 in vitro enhances proliferation and IL-2 receptor expression on lymphocytes, favouring CD8⁺ cells while suppressing the generation of LAK activity. Intravenous administration of Bryostatin increases the potential of IL-2 to induce proliferation and LAK activity in lymphocytes which, taken together with its putative direct antitumour effect, makes Bryostatin an interesting candidate for clinical trials in combination with IL-2.B.F. and P.L.S. are supported by the Cancer Research Campaign     

Colonization of chicks by non-culturable Campylobacter spp.

Six suspensions of non-culturable Campylobacter spp. were administered by gavage to day-of-hatch chicks. Four non-culturable isolates of Campylobacter spp. were found to colonize low numbers (5/79) of 1-week-old chicks, while two isolates did not (0/30). The original and recovered Campylobacter spp. isolates were serotyped and examined by restriction enzyme analysis. Evidence of clonality of two Camp, jejuni isolates was demonstrated.     

Response of Campylobacter jejuni to combinations of ferrous sulphate and cadmium chloride

On Mueller Hinton (MH) agar, Campylobacter jejuni showed 20.0 and 30.9 mm zones of inhibition surrounding discs impregnated with 2.5 and 20 micrograms CdCl2 respectively. The minimal inhibitory concentration (MIC) ranged from 0.64 to 3.2 micrograms CdCl2/ml of MH agar for four C. jejuni strains. In the presence of 23 micrograms FeSO4/ml of MH the MIC increased to a range of 1.5-5.4 micrograms CdCl2/ml of MH. Moreover, the numbers of colonies present on MH supplemented with FeSO4 were greater than on MH without iron. The growth response of C. jejuni in the presence of 0.025% (w/v) FeSO4 in MH broth was increased about 10,000 fold in three of four strains when compared with the growth in unsupplemented MH broth. Zones of inhibition surrounding 20 micrograms discs of CdCl, were 50.6 and 24.4 mm on MH and Campy-BAP media respectively, with cells grown on MH. These results suggest that the blood-containing medium 'neutralized' the biocidal influence of the CdCl2. In comparison, C. jejuni inoculum from fluid thioglycollate (FT) medium showed smaller zones of inhibition. These decreased from 34.9 mm on MH agar to 19.6 mm on Campy-BAP agar, suggesting that components in the FT growth medium ameliorated the toxic influence of CdCl2. Atomic absorption spectroscopy analysis indicated mean values (mg/100 g dry weight) of selected metals bound by C. jejuni as: Cu, 10.4; Mg, 146; Na, 2385; Fe, 45.1; Zn, 13.0; and K, 172.     

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors.

To cast light on the subunit composition of native NMDA-type glutamate receptors, four cloned subunits of the NMDA receptor have been expressed, in pairs, in Xenopus oocytes, and their single-channel properties have been measured. The conductances of the channels, and their characteristic patterns of sublevel transitions, turn out to be useful diagnostic criteria for subunit composition. The NR1-NR2A and NR1-NR2B combinations (which have identical TM2 sequences) are very similar to each other. Both have 50 pS openings and brief 40 pS sublevels (in 1 mM external Ca2+), with similar mean lifetimes and frequencies. They also show close quantitative resemblance to the channels of hippocampal CA1 and dentate gyrus cells and of cerebellar granule cells, except that the NR1-NR2A combination has a lower glycine sensitivity than the native channels. In contrast, the NR1-NR2C combination produces a channel with 36 pS and 19 pS conductances of similar (brief) duration; these closely resemble the 38-18 pS channels that have previously been observed in large cerebellar neurons in culture (together with 50 pS channels).     

Estradiol regulation of secretory component in the uterus of the rat: evidence for involvement of RNA synthesis

The present studies were undertaken to characterize the response of uterine secretory component (SC) to estradiol. Administration of estradiol for 3 days to ovariectomized rats before incubation of uterine tissues resulted in a marked accumulation of SC in the incubation media. When uteri from ovariectomized rats treated with progesterone or testosterone were incubated, very little SC accumulated in the media, indicating that the estradiol-stimulated increase is hormone-specific. When uteri from rats that received estradiol for 6 days were compared with uteri from 3-day treated rats, SC release during a 24-hr incubation period was the same. This finding indicates that in the presence of prolonged estradiol exposure, SC production continues. The estradiol-induced accumulation of SC in culture is not due to the release of pre-formed uterine SC. When tissue SC levels were measured after 3 days of estradiol treatment, very little tissue SC was found relative to that released into culture media during 24 hr of incubation. The addition of actinomycin D to the incubation media markedly inhibited SC release by uteri from estradiol-treated rats. The release of SC was also inhibited by alpha-amanitin, a known inhibitor of Type II polymerase. These studies demonstrate that estradiol stimulation of SC is markedly reduced by inhibitors of RNA synthesis, and suggest that estradiol regulation of SC is mediated through uterine mRNA synthesis.     

Repetitive extragenic palindromic sequences: a major component of the bacterial genome

We describe a remarkably conserved nucleotide sequence, the many copies of which may occupy up to 1% of the genomes of E. coli and S. typhimurium. This sequence, the REP (repetitive extragenic palindromic) sequence, is about 35 nucleotides long, includes an inverted repeat, and can occur singly or in multiple adjacent copies. A possible role for the REP sequences in regulation of gene expression has been thoroughly investigated. While the REP sequences do not appear to modulate differential gene expression within an operon, they can affect the expression of both upstream and downstream genes to a small extent, probably by affecting the rate of mRNA degradation. Possible roles for the REP sequence in mRNA degradation, chromosome structure, and recombination are discussed.