PstB protein of the phosphate-specific transport system of Escherichia coli is an ATPase.

The PstB protein of the phosphate-specific transport (Pst) system of Escherichia coli bound and hydrolyzed ATP, producing ADP. Urea-treated denatured PstB did not bind ATP. The N-terminal amino acid sequence of the immune serum-precipitable PstB protein was determined, and it corresponded to that deduced from the DNA sequence.

Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli.

In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source. The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate. These cells were devoid of alkaline phosphatase activity. However, the phoA promoter was fully active in a glpT mutant. These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium.

Role of PhoU in phosphate transport and alkaline phosphatase regulation.

The negative regulatory function of PhoU in alkaline phosphatase (AP) was suggested by the behavior of K10 phoU35 carrying a missense mutation whose product was detected by immunoblotting. To define more clearly the regulatory function of this protein for the synthesis of AP, we constructed a null mutation. The constitutive synthesis of AP in this phoU deletion strain confirmed the negative role of PhoU. However, the expression of the PhoU protein from an isopropyl-beta-D-thiogalactopyranoside-inducible promoter had no effect on the repression of AP synthesis. Furthermore, the involvement of PhoU in free-Pi uptake was demonstrated. These results provide evidence that PhoU participates in Pi transport and in the regulatory role of the phosphate-specific transport system.

From cell membrane to nucleotides: the phosphate regulon in Escherichia coli.

Most of the essential cellular components, like nucleic acids, lipids and sugars, are phosphorylated. The phosphate equilibrium in Escherichia coli is regulated by the phosphate (Pi) input from the surrounding medium. Some 90 proteins are synthesized at an increased rate during Pi starvation and the global control of the cellular metabolism requires cross-talk with other regulatory mechanisms. Since the Pi concentration is normally low in E. coli's natural habitat, these cells have devised a mechanism for synthesis of about 15 proteins to accomplish two specific functions: transport of Pi and its intracellular regulation. The synthesis of these proteins is controlled by two genes (the phoB-phoR operon), involving both negative and positive functions. PhoR protein is a histidine protein kinase, induced in Pi starvation and is a transmembrane protein. It phosphorylates the regulator protein PhoB which is also Pi starvation-induced. The PhoB phosphorylated form binds specifically to a DNA sequence of 18 nucleotides (the pho Box), which is part of the promoters of the Pho genes. The genes controlled by phoB constitute the Pho regulon. The repression of phoA (the gene encoding alkaline phosphatase) by high Pi concentrations in the medium requires the presence of an intact Pst operon (pstS, pstC, pstA, pstB and phoU) and phoR. The products of pstA and pstC are membrane bound, whereas the product of pstS is periplasmic and PstB and PhoU proteins are cytoplasmic. The function of the PhoU protein may be regulated by cofactor nucleotides and may be involved in signaling the activation of the regulon via PhoR.

Molecular aspects of phosphate transport in Escherichia coli.

Escherichia coli transports inorganic phosphate (Pi) by the low-affinity transport system, Pit. When the level of the external Pi is lower than 20 microM, another transport system, Pst, is induced with a Kt of 0.25 microM. An outer-membrane porin, PhoE, with a Km of about 1 microM is also induced. The outer membrane allows the intake of organic phosphates which are degraded to Pi by phosphatases in the periplasm. The Pi-binding protein will capture the free Pi produced in the periplasm and direct it to the transmembrane channel of the cytoplasmic membrane. The channel consists of two proteins, PstA and PstC, which have six and five transmembrane helices, respectively. On the cytoplasmic side of the membrane the channel is linked to the PstB protein, which carries a nucleotide (probably ATP)-binding site. PstB probably provides the energy required by the channel to free Pi. The Pst system has two functions in E. coli: (i) the transport of Pi, and (ii) the negative regulation of the phosphate regulon (a complex of 20 proteins mostly related to organic phosphate transport). It is remarkable that these two functions are not related, since the repressibility of the regulon depends on the integral structure of Pst (PiBP + PstA + PstC + PstB) and not on the Pi transported. Another gene of the pst operon, phoU, produces a protein involved in the negative regulation of the Pho regulon, but the mechanism of this function has not been explained. Thus the regulatory function of the Pst system remains obscure. Its basal level, present when Pi is abundant, is sufficient to repress the Pho regulon but the negative regulatory function is lost upon Pi starvation.

Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins.

We observed that wild-type Escherichia coli utilized a linear polyphosphate with a chain length of 100 phosphate residues (poly-P100) as the sole source of phosphate in growth medium. A mutation in the gene phoA of alkaline phosphatase or phoB, the positive regulatory gene, prevented growth in this medium. Since no alkaline phosphatase activity was detected outside the wild-type cells, the periplasmic presence of the enzyme was necessary for the degradation of polyphosphate. A 90% reduction in the activity of periplasmic acid phosphatase with a pH optimum of 2.5 (delta appA mutants) did not affect polyphosphate utilization. Of the porins analyzed (OmpC, OmpF, and PhoE), the phoB-inducible porin PhoE was not essential since its absence did not prevent growth. To study how poly-P100 diffused into the cells, we used high-resolution 31P nuclear magnetic resonance (31P NMR) spectroscopy. The results suggest that poly-P100 entered the periplasm and remained in equilibrium between the periplasm and the medium. When present individually, porins PhoE and OmpF facilitated a higher permeability for poly-P100 than porin OmpC did. The degradation of polyphosphate by intact cells of E. coli observed by 31P NMR showed a time-dependent increase in cellular phosphate and a decrease in polyphosphate concentration.

The influence of thyroid hormones on colony growth of peripheral CFU-GM from normal and leukemic subjects.

In order to clarify the alterations of granuloblastic cells in chronic and acute myeloid leukemia, the colony growth behavior of cultured CFU-GM from the peripheral blood of normal and leukemic subjects was examined in basal conditions and after adding to the medium T3 or T4 and/or thioproline and/or flurbiprofen. These drugs had in previous investigations proved their ability to modify cellular receptors and the uptake of thyroid hormones. The study was carried out in semisolid (double agar layer) and liquid medium, utilizing the techniques described previously. Both thyroid hormones enhanced the colony growth from normal peripheral blood CFU-GM and the response was more evident with T4 than T3. The effect on leukemic CFU-GM (from CML and AML) was less clear, probably due to the presence in leukemic cells of a defect of cellular uptake and to the utilization of T3 and T4. Indeed, on addition to the culture medium of thioproline, which modifies membrane permeability, and of fluorbiprofen, which inhibits PGE synthesis, the colony number and growth from leukemic CFU increased considerably in accordance with the results of our previous studies on these substances showing that they are able to modify cellular receptors for thyroid and several other hormones.

[Penbutolol and arterial hypertension]. / Penbutololo e ipertensione arteriosa.

Penbutolol has proved particularly effective and suitable for the treatment, even on a long-term basis, of recently developed hypertension, especially in its hyperkinetic forms. The drug produces minimal side effects, is well tolerated and gives an early therapeutic response. In addition penbutolol does not cause any significant alterations in the biohumoral parameters of the patients treated and is ideal for combination with dihydralazine, reserpine and dihydrochlorotiazide in the treatment of more stubborn cases, making it possible to reduce the doses of the other drugs without causing bradycardia.

Nucleotide pool in pho regulon mutants and alkaline phosphatase synthesis in Escherichia coli.

The intracellular nucleotide pool of Escherichia coli W3110 reproducibly changes from conditions of growth in phosphate excess to phosphate starvation, with at least two nucleotides appearing under starvation conditions and two nucleotides appearing only under excess phosphate conditions. Strains bearing a deletion of the phoA gene show the same pattern, indicating that dephosphorylation by alkaline phosphatase is not responsible for the changes. Strains with mutations in the phoU gene, which result in constitutive expression of the pho regulon, show the nucleotide pattern of phosphate-starved cells even during phosphate excess growth. These changes in nucleotides are therefore due to phoU mutation but not to alkaline phosphatase constitutivity. In fact, a phoR (phoR68) mutant strain has the patterns of the wild type in spite of being constitutive for alkaline phosphatase. That these nucleotides might be specific signals for pho regulon expression was supported by the fact that the two nucleotides appearing under phosphate starvation induced the synthesis of alkaline phosphatase in repressed permeabilized wild-type cells under conditions of phosphate excess.

Amount and chain length of polyphosphates in Escherichia coli depend on cell growth conditions.

Anaerobiosis induced an accumulation of polyphosphates (poly Pi) in a phosphate-rich medium by an alkaline-phosphatase constitutive mutant of Escherichia coli. The total poly Pi content was maximum at around 6 h of anaerobic growth. Both trichloroacetic acid- and NaOH-soluble poly Pi were found to be present. The acid-soluble fraction consisted mainly of a linear polymer of about 20 +/- 5 phosphate units, whereas the alkali-extractable poly Pi fraction contained at least four molecular species of higher chain length as determined by gel filtration. The majority of poly Pi extracted at 6 h had lower chain lengths than those extracted from cells incubated for 24 h. In vivo 31P nuclear magnetic resonance spectra of E. coli cells as a function of growth conditions were consistent with the in vitro extract results.

[The role of plasmapheresis in the treatment of primary and secondary cryoglobulinemia]. / Ruolo della plasmaferesi nel trattamento delle crioglobulinemie primarie e secondarie.

The data on monoclonal or mixed cryoglobulinaemia patients admitted to Pavia University's 1st Medical Division since 1970 were examined. Complications included peripheral microangiopathies caused by immune complexes and especially kidney lesions of varying severity caused by immune complexes precipitated in the basal membrane. Although the course of the condition was ameliorated in all patients given immunosuppressive treatment alone, this could not prevent the appearance of progressively worsening kidney conditions. Terminating in severe renal failure these were often complicated by encephalopathies, disturbed microcirculation in the brain and hypertension patients given plasmapheresis at an early stage, at the first sign of renal or brain circulation problems, responded brilliantly with regression of all clinical symptoms. Where plasmapheresis was not given until an advanced stage of the kidney disease, a satisfactory improvement was obtained and the quality and expectancy of life enhanced, even though a total cure was not achieved. Regular cycles of plasmapheresis indubitably protect the patient from irreversible renal or microvascular conditions so that immunosuppressive treatment can effectively control the cryoglobulinaemia.

Identification of the phoM gene product and its regulation in Escherichia coli K-12.

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.

Drugs affecting the hormonal receptors of normal and leukaemic peripheral leucocytes.

The authors investigated the behaviour of steroid hormone uptake in leukaemic cells (CML, CLL, AML, ALL), in basal conditions and after incubation with drugs which modify the cellular concentration of cAMP, PGE and PGF. The results demonstrated the presence in leukaemic cells of an alteration in the incorporation of steroid hormones. This alteration was scarcely modified by incubation with theophylline, which increases cellular concentration of cAMP. On the other hand, it was moderately counteracted by thioproline and was evidently inhibited by flurbiprofen, which also reduced cellular concentrations of prostaglandins, particularly PGE2, with the exception of PGF2 which showed a poor response. Differences were observed in the behavior of hormonal uptake of CML, in contrast to that of AML, CLL and ALL peripheral leucocytes.

[Plasmapheresis and survival of patients with myeloma and secondary renal insufficiency]. / Plasmaferesi e sopravvivenza del paziente mielomatoso con insufficienza renale secondaria.

The mortality rate due to renal insufficiency in patients with myelomas was studied and the results obtained compared with figures on patients given plasmapheresis to correct the insufficiency. The results of the comparison confirm the value of the immediate use of plasmapheresis on patients with myelomas. In a large percentage of cases, the technique either completely cured or significantly improved renal dysfunction, thus normalizing the blood and urine situation and improving the patients' chances of survival.

[Behavior of the principal protein hormones in normal and leukemic leukocytes]. / Comportamento dei principali ormoni proteici nei leucociti normali e leucemici.

GH, LH, insulin and glucagon patterns were studied in the peripheral leukocytes of normal subjects (granulocytes and lymphocytes separated on a Ficoll-Hypaque gradient) and leukaemic patients (CML, AML, CLL, and ALL), using a double antibody RIA on whole cells. The uptake of 125I-labelled insulin and GH by these cells was also assessed. The results showed that in leukaemia, particularly CLL, ALL and AML, though not in CML, there was a constant reduction in hormone values, plus depressed GH and insulin uptake. The only exceptions were glucagon and insulin in CML, and LH in CLL, since their concentrations were normal or clearly enhanced. The data are seen as an expression of a membrane receptor block extending to several hormones with structural differences (protein, steroid, T3 and T4), capable of altering the ability of leukaemic cells to respond to ordinary factors modulating their differentiation, functional activity, and the expansion of the corresponding stem cell compartment.

Seminalplasmin inhibits transcription and translation of phi80 DNA in vitro.

Seminalplasmin, an antimicrobial protein from bovine seminal plasma that has been earlier shown to inhibit transcription in whole cells and by purified RNA polymerase in vitro, but not translation in whole cells, is now shown to inhibit both transcription and translation independently of each other, in a coupled transcription-translation system from E. coli using phi80dphoAlacZ DNA as the template.

Genetic and physiological tests of three phosphate-specific transport mutants of Escherichia coli.

Phosphate-specific transport system mutations phoT35, pst-2, and phoS25-(Am) were mapped between bgl and glmS, at about 83 min on the Escherichia coli chromosome. All three mutations were recessive to wild-type genes on transducing bacteriophage lambda asn. The phoS25 (Am) and pst-2 mutations were also recessive to transducing phage lambda dglm; however, the phoT35 mutation was not. This suggests that phoT35 lies in a different complementation group from phoS25 (Am) or pst-2. Isogenic series of strains carrying these mutations were constructed in two genetic backgrounds, pit+ (wild type) and pit (relying entirely on the phosphate-specific transport system for phosphate uptake). The pst-2 pit double mutant was incapable of Pi utilization, but the phoT35 pit double mutant exhibited no such deficiency.

Pleiotropic effects of alkaline phosphatase regulatory mutations phoB and phoT on anaerobic growth of and polyphosphate synthesis in Escherichia coli.

The appearance during anaerobiosis of spontaneous phoT phoB double mutants in a phoT background is described. During both exponential growth and stationary phase, selection against the phoT mutants relative to the wild type was evident. This reduction in viability of phoT mutants was suppressed in phoT phoB double mutants. Sensitivity to anaerobiosis was shown to be correlated with polyphosphate overproduction. A possible pleiotropic function of phoT and phoB is suggested.