Synthesis of a new element with atomic number Z = 117.

The discovery of a new chemical element with atomic number Z=117 is reported. The isotopes (293)117 and (294)117 were produced in fusion reactions between (48)Ca and (249)Bk. Decay chains involving 11 new nuclei were identified by means of the Dubna gas-filled recoil separator. The measured decay properties show a strong rise of stability for heavier isotopes with Z > or = 111, validating the concept of the long sought island of enhanced stability for superheavy nuclei.

Identification of a candidate membrane protein for the basolateral peptide transporter of rat small intestine.

A candidate protein for the basolateral peptide transporter of rat jejunum is described. Vascular perfusion of the photoaffinity label, [4-azido-D-phe]-L-ala (2.5mM), had no effect on the transepithelial transport of the non-hydrolysable dipeptide D-phe-L-gln (1mM) from the lumen, its mucosal accumulation or wash-out into the vascular perfusate. When the label was perfused luminally, the transepithelial transport of D-phe-L-gln was inhibited by 38% (P<0.001) and accumulation increased by 62% (P<0.05). These data are consistent with those of a basolateral transporter that is strongly asymmetric in its substrate binding and transport properties. Labelling of basolateral membrane vesicles with [4-azido-3,5-3H-D-phe]-L-ala revealed that the majority of label was incorporated into a single protein of M(r)112+/-2 kDa and pI 6.5. MALDI-TOF analysis of tryptic digests of the protein followed by database searches established that this protein was novel with no obvious similarity to PepT1, the apical membrane transporter.

Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1.

The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N-acetyl-Phe (Ac-Phe), phe-amide (Phe-NH2), N-acetyl-Phe-amide (Ac-Phe-NH2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [3H]-D-Phe-L-Gln. In an equivalent set of experiments, the blocked peptides Ac-Phe-Tyr, Phe-Tyr-NH2 and Ac-Phe-Tyr-NH2 were compared with the parent compound Phe-Tyr. Comparing amino acids and derivatives, only Ac-Phe was an effective inhibitor of peptide uptake (Ki = 1.81+/- 0.37 mM). Ac-Phe-NH2 had a very weak interaction with PepT1 (Ki = 16.8+/-5.64 mM); neither Phe nor Phe-NH2 interacted with PepT1 with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (Ki = 0.10+/-0.04 mM). The blocked C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relatively high affinity (Ki = 0.94+/-0.38 mM). Both Ac-Phe-Tyr and Ac-Phe-Tyr-NH2 interacted weakly with PepT1 (Ki = 8.41+/-0.11 and 9.97+/-4.01 mM, respectively). The results suggest that the N-terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C-terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.

Peptide mimics as substrates for the intestinal peptide transporter.

4-Aminophenylacetic acid (4-APAA), a peptide mimic lacking a peptide bond, has been shown to interact with a proton-coupled oligopeptide transporter using a number of different experimental approaches. In addition to inhibiting transport of labeled peptides, these studies show that 4-APAA is itself translocated. 4-APAA transport across the rat intact intestine was stimulated 18-fold by luminal acidification (to pH 6.8) as determined by high performance liquid chromatography (HPLC); in enterocytes isolated from mouse small intestine the intracellular pH was reduced on application of 4-APAA, as shown fluorimetrically with the pH indicator carboxy-SNARF; 4-APAA trans-stimulated radiolabeled peptide transport in brush-border membrane vesicles isolated from rat renal cortex; and in Xenopus oocytes expressing PepT1, 4-APAA produced trans-stimulation of radiolabeled peptide efflux, and as determined by HPLC, was a substrate for translocation by this transporter. These results with 4-APAA show for the first time that the presence of a peptide bond is not a requirement for rapid translocation through the proton-linked oligopeptide transporter (PepT1). Further investigation will be needed to determine the minimal structural requirements for a molecule to be a substrate for this transporter.

4-aminomethylbenzoic acid is a non-translocated competitive inhibitor of the epithelial peptide transporter PepT1.

1. 4-Aminomethylbenzoic acid, a molecule which mimics the special configuration of a dipeptide, competitively inhibits peptide influx in both Xenopus Laevis oocytes expressing rabbit PepT1 and through PepT1 in rat renal brush border membrane vesicles. 2. This molecule is not translocated through PepT1 as measured both by direct HPLC analysis in PepT1-exp ressing oocytes and indirectly by its failure to trans-stimulate labelle d peptide efflux through PepT1 in oocytes and in renal membrane vessicle s. 3. However 4-aminiomethylbenzoic acid does reverse trans-stimulation through expressed PepT1 of labelled peptid efflux induced by unlabelled peptide. Quantitatively this reversal is compatible with 4-aminomethyl benzoic acid competitively binding to the external surface of PepT1. 4. 4-Aminomethylbenzoic acid (the first molecule discovered to be a non-translocated competitive inhibitor of proton-coupled oligopeptide transport) and its derivatives may thus be particularly useful as experimental tools.

The influence of luminal pH on transport of neutral and charged dipeptides by rat small intestine, in vitro.

Four hydrolysis-resistant dipeptides (D-phenylalanyl-L-alanine, D-phenylalanyl-L-glutamine, D-phenylalanyl-L-glutamate and D-phenylalanyl-L-lysine) were synthesized to investigate the effects of net charge on transmural dipeptide transport by isolated jejunal loops of rat small intestine. At a luminal pH of 7.4 and a concentration of 1 mM the two dipeptides with a net charge of -1 and +1 were transported at substantially slower rates (18 +/- 1.3 and 8.4 +/- 1.3 nmol min(-1)(g dry wt.)(-1), respectively) than neutral D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine (87 +/- 0.2 and 197 +/- 14 nmol min(-1)(g dry wt.)(-1), respectively). We investigated the effects of luminal pH on dipeptide transport by varying the NaHCO3 content of Krebs Ringer perfusate equilibrated with 95% 02/5% CO2. The pH changes did not affect water transport, but serosal glucose appearance increased significantly at pH 6.8. Transmural transport of D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine at pH 6.8 was stimulated (P < 0.01) by 61% and 49%, respectively, whereas the lower pH increased the rate for negatively charged D-phenylalanyl-L-glutamate by 306% (P < 0.01) and decreased that for positively charged D-phenylalanyl-L-lysine by 46% (P < 0.05). Increasing luminal pH to 8.0 inhibited D-phenylalanyl-L-alanine transport by 60%, whereas D-phenylalanyl-L-lysine transport was 60% faster.

A model for the kinetics of neutral and anionic dipeptide-proton cotransport by the apical membrane of rat kidney cortex.

1. Kinetics of influx (mediated through peptide-proton cotransport) of two labelled dipeptides has been studied in apical membrane vesicles isolated from rat renal cortex. The substrates (neutral D-Phe-L-Ala and anionic D-Phe-L-Glu) have previously been shown to be transported through a single system but with different stoichiometry of proton coupling. 2. The initial rate of influx of both peptides was determined under a set of defined conditions allowing extravesicular pH, intravesicular pH, transmembrane pH and membrane potential (Em) to be varied systemically and independently. From this data the kinetic constants K(m) and Vmax were derived for each condition. Very substantial effects of pH, pH gradient and membrane potential were found; there were consistent quantitative differences when the substrates were compared. 3. Efflux of the two peptides from preloaded vesicles was also determined. At pH 5.5 (intra- and extravesicular), but not at pH 7.4, the rate constants for efflux of the two peptides were similar and addition to the extravesicular medium of unlabelled D-Phe-L-Glu (but not D-Phe-L-Ala) trans-stimulated efflux of both peptides to a similar extent; the extent of this trans-stimulation was insensitive to alterations in membrane potential. 4. A model based on a combination of classical carrier theory (the carrier being negatively charged) and of two sequential protonation steps (both to external sites predicted to be in the membrane electrical field) is described. Qualitatively this adequately accounts for all the observations made and allows for the dependence of the stoichiometry of proton-peptide coupling on the net charge carried by the substrate. Quantitatively a 50-fold greater rate of reorientation of the free carrier when unprotonated is predicted to be responsible for the coupling of proton and peptide transport. 5. Our results and the model are discussed with respect to the recently elucidated primary structure of mammalian peptide transporters.

Substrate-charge dependence of stoichiometry shows membrane potential is the driving force for proton-peptide cotransport in rat renal cortex.

The proton dependence of the transport of three labelled, hydrolysis-resistant synthetic dipeptides carrying a net charge of -1, 0 or +1 has been investigated in a brush border membrane vesicle preparation obtained from rat renal cortex. Cross-inhibition studies are consistent with the transport of all peptides studied being through a single system. The extent and time course of uptake in response to an inwardly directed electrochemical gradient of protons differed for each peptide. For the cationic peptide D-Phe-L-Lys this gradient did not stimulate the initial rate of uptake, while for the neutral dipeptide D-Phe-L-Ala and the anionic peptide D-Phe-L-Glu stimulation was observed. However, the effect on D-Phe-L-Glu was more marked than that on D-Phe-L-Ala and the proton activation differed for these two peptides. The calculated Hill coefficients for the two proton-dependent peptides were 1.14 +/- 0.16 and 2.15 +/- 0.10 for D-Phe-L-Ala and D-Phe-L-Glu, respectively, providing evidence that the stoichiometry of proton:peptide cotransport is different for each peptide (0:1, 1:1 and 2:1 for D-Phe-L-Lys, D-Phe-L-Ala and D-Phe-L-Glu respectively); studies on energetics are compatible with this conclusion. The physiological and molecular implications of this model are discussed, as are the applicability of the conclusions to secondary active transport systems more generally.

Dipeptide transport and hydrolysis in rat small intestine, in vitro.

A range of natural and mixed D-/L-stereoisomer phenylalanine dipeptides was used to investigate peptide uptake and hydrolysis by isolated rings of rat jejunum. Characterisation of dipeptide hydrolysis by the brush border fraction revealed apparent Km values in the 0.1-1.0 mM range which, except for the charged dipeptides, were significantly higher than those for hydrolysis by the cytosolic fraction. Uptake of L-/L-dipeptides into jejunal rings, which was followed by HPLC, was unaffected by the presence of peptidase inhibitors in the incubation medium although the absorbed peptides were completely hydrolysed in the cytosol; comparison of the effects of excess leucine on dipeptide uptake and on the uptake of the two constituent amino acids were also consistent with absorption of intact dipeptide followed by cytosolic hydrolysis. The uptake of hydrolysis-resistant mixed D-/L-dipeptides was also studied and confirmed that peptide uptake preceded hydrolysis; D-alanyl-L-phenylalanine accumulated within the rings to twice the medium concentration.

Dipeptide transport and hydrolysis in isolated loops of rat small intestine: effects of stereospecificity.

1. Isolated jejunal loops of rat small intestine were perfused by a single pass of bicarbonate Krebs-Ringer solution containing either D- or L-phenylalanine or one of eight dipeptides formed from D- or L-alanine plus D- or L-phenylalanine. 2. At 0.5 mM L-phenylalanyl-L-alanine increased serosal phenylalanine appearance to forty times the control rate giving a value similar to that found with 0.5 mM free L-phenylalanine. No serosal dipeptide could be detected. 3. Perfusions with the two mixed dipeptides with N-terminal D-amino acids (D-alanyl-L-phenylalanine and D-phenylalanyl-L-alanine) gave rise to the appearance of intact dipeptides in the serosal secretions although there were substantial differences in their rates of absorption and subsequent hydrolysis. 4. L-Alanyl-D-phenylalanine was absorbed from the lumen three to five times as fast as L-phenylalanyl-D-alanine. At 1 mM L-alanyl-D-phenylalanine transferred D-phenylalanine across the epithelial layer at more than seven times the rate found with the same concentration of the free D-amino acid. 5. Perfusions with D-alanyl-D-phenylalanine or D-phenylalanyl-D-alanine showed that these two dipeptides are poor substrates for both transport and hydrolysis by the rat small intestine. 6. Analysis of mucosal tissue extracts after perfusion with the two mixed dipeptides with N-terminal D-amino acids revealed that both dipeptides were accumulated within the mucosa and suggested that exit across the basolateral membrane was rate limiting for transepithelial dipeptide transport.

Tripeptide transport in rat lung.

Transport of L-alanyl-D-phenylalanyl-L-alanine was investigated with an in situ vascular perfusion preparation of rat lung and brush border membrane vesicles prepared from type II pneumocytes. In the perfused lung 1 mM tripeptide was transported intact from the alveolar lumen to the vascular perfusate at a mean rate of 25.1 +/- 1.29 (3) nmol/min per g dry weight. D-Phenylalanine also appeared in the vascular perfusate at a rate of 21.9 +/- 1.74 (3) nmol/min per g dry weight indicating that 47% of the absorbed tripeptide was split during passage across the epithelial layer. No dipeptide could be detected in the vascular effluent during perfusions with tripeptide. Rapid L-alanyl-D-phenylalanyl-L-alanine uptake occurred with fresh apical membrane vesicles prepared from type II pneumocytes and this was abolished by treatment with 0.1% triton. The related tripeptide, D-alanyl-L-phenylalanyl-D-alanine, was taken up significantly more slowly by the vesicles. D-phenylalanyl-L-alanine and D-phenylalanyl-D-alanine, were also studied with the vascularly perfused preparation; the mixed dipeptide appeared in the vascular perfusate significantly faster than L-alanyl-D-phenylalanyl-L-alanine whereas D-phenylalanyl-D-alanine appeared more slowly and was not hydrolysed.

Normal cellular Ha ras p21 protein causes local disruption of bilayer phospholipid.

We have investigated the interactions of the p21 protein of c-Ha ras with its phospholipid environment. Gel filtration of detergent-"solubilized" p21 revealed that this preparation consisted of a mixture of multimolecular aggregates of protein and phospholipid and also a population of individual p21 molecules. Addition of 8 M urea to p21 preparations increased the solubility of the molecule in detergent solutions upon the removal of this denaturant. The progressive addition of the detergent cholate appeared to increase the efficiency of p21 preparations to bind GTP. This affinity for GTP was not removed even at high detergent concentrations, when delipification of the p21 was presumably effected. Modification of the composition of the phospholipid species surrounding the protein did not appear to alter its affinity for GTP. Electron spin resonance studies with membrane spin-labels indicated a perturbation of the bilayer extending to between 44 and 100 phospholipids surrounding the molecule. However, no evidence was found for any population of intimately bound phospholipid, which is seen as an annulus of about 30 lipids in transmembrane proteins such as Ca2+-ATPase. From these results we propose that the Ha ras p21 protein has the ability to associate directly with the membrane in a manner clearly discernible from that of a transmembrane protein.

Detection of herpes simplex virus type 2-specific antibody with glycoprotein G.

A recently described herpes simplex virus (HSV) type 2 (HSV-2)-specific glycoprotein (gG-2) was purified on an immunoaffinity column prepared with monoclonal antibody. This purified antigen was used in an immunodot enzymatic assay on nitrocellulose paper for the detection of HSV-2 antibodies in human serum. The test was very sensitive in that HSV-2 antibodies were detected in the convalescent sera of 132 of 134 patients with recurrent genital infections in which HSV-2 had been isolated earlier. Antibodies to gG-2 were detected in 17% of sera obtained within 10 days after the onset of a primary HSV infection and in 95% of sera obtained more than 10 days after onset. The specificity of the immunodot assay was demonstrated by testing sera from 245 HSV-seronegative adults, 344 children, 29 nuns, and 13 patients with primary genital HSV-1 infections. None of these 631 sera was reactive with the gG-2 antigen. When compared with a microneutralization test, the immunodot assay was found to be more specific in detecting HSV-2 antibodies. Reproducibility of the gG-2 assay, obtained by retesting 391 sera, was 95%. Thus, this assay has the sensitivity, specificity, and reproducibility necessary for the measurement of HSV-2 antibodies in seroepidemiological studies.

[7-Methyltryptophan9]-beta-corticotropin-(1-24): a hyperactive Synacthen analogue.

[7-Methyltryptophan9]-beta-corticotropin-(1-24) has been synthesised. In an isolated adrenal cell bioassay, it has 2.7 times the steroidogenic activity of beta-corticotropin-(1-24) (Synacthen).

ELISA for the detection of herpes simplex virus antigens in the cerebrospinal fluid of patients with encephalitis.

An inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of herpes simplex virus antigens in cerebrospinal fluid (CSF) has been developed. A Triton X-100 extract of herpes simplex virus type 1 (HSV-1) infected HEp-2 cells was used to coat wells of polyvinyl chloride plates. Rabbit anti-HSV-1 globulin served as the reference antibody and the CSF specimens were tested at a final dilution of 1:4. Positive results were obtained in CSF specimens from 11/18 (61%) neonates with HSV infection, 15/23 (65%) older individuals with HSV culture positive brain biopsies, and in 4/29 (14%) patients with culture negative brain biopsies. The assay was negative with CSF from 14 infants without HSV infections, from 30 patients with bacterial meningitis and 10 with cryptococcal meningitis. The test was positive in 10/21 patients within 10 days of onset, 11/14 within 11-20 days, and in 5/6 more than 20 days after onset of the herpetic infection. The overall sensitivity of the assay was 63% and the specificity was 95%.

Determination of herpes simplex virus type-specific antibodies by enzyme-linked immunosorbent assay.

We determined type-specific antibodies to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by an indirect enzyme-linked immunosorbent assay, using as antigens HSV-1 glycoprotein gC-1 and a HSV-2-specific polypeptide purified on affinity columns of monoclonal antibodies. All sera were initially screened for HSV antibodies by the enzyme-linked immunosorbent assay with a pool of Triton X-100-extracted antigens of HSV-1- and HSV-2-infected HEp-2 cells. The titer of HSV antibodies was predicted from a linear regression curve based on the absorbance of the initial 1:50 serum dilution. The sensitivity and specificity of the screening assay and of the assay for type-specific antibodies were established.

Electron microscopy in the routine screening of newborns with congenital cytomegalovirus infection.

The pseudoreplica method of electron microscopy (EM) was evaluated as a rapid screening technique for the detection of cytomegaloviruria in 3056 neonates in a predominantly lower socioeconomic population. Virus isolation methods detected 49 (1.6%) CMV-positive individuals. When pools of three to five urines were tested, 26 (54%) of the culture-positive neonates were identified by EM; however, testing of individual urines increased EM detection to 33 (67%). Almost all of these urines, as well as urine or oral specimens obtained on follow-up visits, which had infectivity titers greater than or equal to 10(4)/ml were EM-positive, whereas only half of the specimens with titers less than 10(4)/ml were EM-positive. All the symptomatic neonates were detected by EM, suggesting that electron microscopy would be most valuable as a diagnostic aid in this group of CMV-infected neonates.