In vivo experimental modelling of the infectious process caused by stable L forms of the causative agent of typhoid

To simulate the infectious process and to study the persistence of L-forms, rabbits and guinea pigs were infected with S. typhi stable L-forms. The materials presented in this work indicate that both subconjunctival and intraperitoneal infection led to the development of the clinically indistinct, but morphologically pronounced pathological process with characteristic localization and typical changes in the gastrointestinal tract. The typical features of the process were the generalized immunomorphological reaction of the lymphoid apparatus with the appearance of light-colored reticulomacrophagal elements, the signs of the activation of humoral and cell-mediated immunity and the formation of small epitheloidocellular granulomas. The results of the investigation indicate that the stable cultures of S. typhi L-forms are highly pathogenic and capable of inducing the infectious process in experimental animals.     

Potassium depletion selectively inhibits sustained diacylglycerol formation from phosphatidylinositol in angiotensin II-stimulated, cultured vascular smooth muscle cells

Potassium depletion decreases blood pressure in vivo and blunts the pressor response to angiotensin II (ang II) without down-regulating the receptor. In cultured rat aortic smooth muscle cells, the ang II-induced signaling sequence is biphasic with rapid hydrolysis of the polyphosphoinositides producing an early (15 s) diacylglycerol (DG) peak and a transient rise in inositol trisphosphate (IP3) and more delayed phosphatidylinositol (PI) hydrolysis resulting in sustained DG formation (peak at 5 min). Exposure of intact vascular smooth muscle cells to low potassium growth medium for 24 h or acutely potassium-depleting cells with nigericin causes selective, marked inhibition of late DG formation (5-min peak inhibited by 60 +/- 8% and 84 +/- 7%, respectively). The early cell response, namely polyphosphoinositide hydrolysis, inositol bis- and trisphosphate production and the 15-s DG peak, is not affected. Analysis of 125I-ang II-binding data reveals no significant differences in either receptor number or binding affinity (Kd) in potassium-depleted cells. Together with its marked inhibitory effect on sustained ang II-induced DG formation, acute potassium depletion effectively blocks internalization of 125I-ang II: there is no significant internalization of the ligand after 5 min at 37 degrees C versus 64 +/- 7% internalization in control cells. Thus, potassium depletion does not alter ang II binding or initial membrane signaling in rat aortic smooth muscle but blocks ligand internalization and selectively and markedly inhibits the development of direct PI hydrolysis and sustained diacylglycerol formation. These findings suggest a role for ligand-receptor processing in generating the sustained cell response and potentially explain the lower blood pressure and decreased pressor response to ang II seen in hypokalemic states in vivo. Furthermore, the ability of K+ depletion to alter secondary signal generation may provide insight into the mechanisms underlying the K+ dependence of a variety of cell functions.     

Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.     

S100 alpha, CAPL, and CACY: molecular cloning and expression analysis of three calcium-binding proteins from human heart.

Elevated levels of intracellular calcium are a major cause of myocardial dysfunction. To find possible mediators of the deregulated calcium we searched for EF-hand calcium-binding proteins of the S100 family. By PCR technology we identified three members of the S100 protein family (S100 alpha, CACY, and CAPL) in the human heart. We cloned the corresponding cDNAs and examined their expression levels in various human tissues by Northern blot analysis. All three proteins are expressed at high levels in the human heart. Whereas CACY and CAPL mRNAs are expressed ubiquitously, S100 alpha mRNA is restricted to heart, skeletal muscle, and brain. Interestingly, the expression pattern of S100 alpha, CACY, and CAPL in human tissues differs significantly from that in rodent tissues.     

Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon

Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the beta-galactosidase gene, lacZ. Rates of beta-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -C are transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nirB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, -D and -C open reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the nirC gene. No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.