The gene encoding BLIP, a beta-lactamase-inhibitory protein, was disrupted in wild-type Streptomyces clavuligerus and in a clavulanic acid non-producing mutant. The resulting BLIP mutant and BLIP/clavulanic acid double mutant showed no residual proteinaceous β-lactamase-inhibitory activity, indicating that only a single β-lactamase-inhibitory protein exists in S. clavuligerus. The lack of any proteinaceous β-lactamase-inhibitory activity in the bli and bli/claR mutants also indicates that BLP, the BLIP-like protein, encoded by S.clavuligerus does not possess β-lactamase-inhibitory activity despite its similarity to BLIP. The bli mutant and the bli/claR double mutant did not show any aberrant growth morphology, sporulation defects, or alterations in cephamycin C production or penicillin G resistance when compared to wildtype S. clavuligerus or to the claR single mutant. Mutants bearing the bli gene disruption did show an elevated level of production of clavam-2-carboxylate and hydroxymethyl clavam as well as clavulanic acid. This phenomenon was observed in the middle stages of production of these clavams but was not detected during maximum production. The production of BLIP was also determined to be down-regulated in a ccaR mutant, lacking the pathwayspecific transcriptional regulator required for production of cephamycin C and clavulanic acid. Sequencing of the regions flanking the bli gene showed the presence of a partial open reading frame that encodes a DNA-binding protein, and several open reading frames apparently involved in the production of an ABC transporter.

Isolated perfused right atria were prepared from virgin cycling rats and from rats at 7, 14, and 21 days of pregnancy. Intraluminal pressure was raised from the basal control level (atmospheric) to 4, 6, and 10 cmH2O. Basal and stretch-induced release of atrial natriuretic factor (ANF) into the perfusate was measured by radioimmunoassay. When atria derived from unmated and from 7-day pregnant rats were distended, mean ANF secretion into the perfusion medium increased by 64 +/- 16 and 89 +/- 31 pg/ml at the highest distending pressure of 10 mmHg. (Mean basal secretion for the two groups was 196 +/- 104 and 181 +/- 63 pg/ml, respectively.) However, at 14 and 21 days, distension failed to elicit any significant increase in ANF release. There was no significant difference between the groups with respect to basal secretion of ANF, nor did pregnancy influence atrial compliance or volume. The slopes of the pressure-volume curves for atria derived from virgin, 7-, 14-, and 21-day-pregnant rats were 0.76 +/- 0.17, 0.623 +/- 0.178, 0.811 +/- 0.177, and 1.050 +/- 0.173, respectively. These results are in agreement with our findings in vivo that plasma ANF levels are elevated at 7 days of pregnancy but that, despite the progressive increase in intravascular volume, they decrease to control levels at 14 and 21 days. Our latest data suggest that this fall is a result of changes in the secretory characteristics of the atrial tissue itself and not necessarily of pregnancy-induced changes in atrial filling.

No abstract