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Resistance to antibiotics is a natural phenomenon that has been noted since the introduction of penicillin in the 1940s 1 . Whenever clinically relevant resistance has emerged, the problem has been tackled with modification of existing antibiotic classes with limited crossresistance to existing drugs or introduction of new classes 2 . The relative ease of the early antibiotic discovery programmes and the financial rewards that followed created a wasteful and uncritical use of antibiotics without adequate consideration of the societal consequences 3 . After this 'golden antibiotic era' , large pharmaceutical companies faced major scientific challenges searching for new antibiotics, especially with regard to penetration barriers and efflux mechanisms in Gram-negative bacteria requiring high antibiotic doses with potential associated toxicity issues 4 . These companies finally abandoned antibacterial drug discovery activities beginning in the 1980s. Furthermore, they lost interest in a field that did not promise ever-increasing market growth and profits. Exits by large pharmaceutical companies have caused concern among scientists, the health-care community, civil society advocates and policymakers Because of the long timelines for research and development, urgently needed responses and action can be calibrated only by knowing the global activities (and lack of activities) in antibacterial drug development. This mapping activity was initiated by the WHO with a recently published global clinical antibacterial pipeline report In contrast to the clinical pipeline, less is known about the preclinical antibacterial pipeline. In this Review, we analyse the preclinical antibacterial pipeline and provide a current snapshot and decision support for all actors in this field and some information on the broader context.

Journal Article Survival models for heterogeneous populations derived from stable distributions Get access PHILIP HOUGAARD PHILIP HOUGAARD Biostatistical Department, Novo Research InstituteNovo Allé, DK-2880 Bagsvœrd, Denmark Search for other works by this author on: Oxford Academic Google Scholar Biometrika, Volume 73, Issue 2, August 1986, Pages 387–396, https://doi.org/10.1093/biomet/73.2.387 Published: 01 August 1986 Article history Received: 01 October 1984 Revision received: 01 December 1985 Published: 01 August 1986

Journal Article A class of multivanate failure time distributions Get access PHILIP HOUGAARD PHILIP HOUGAARD Biostatistical Department, Novo Research InstituteDK-2880 Bagsvaerd, Denmark Search for other works by this author on: Oxford Academic Google Scholar Biometrika, Volume 73, Issue 3, December 1986, Pages 671–678, https://doi.org/10.1093/biomet/73.3.671 Published: 01 December 1986 Article history Received: 01 January 1986 Revision received: 01 May 1986 Published: 01 December 1986

Diabetic nephropathy is the main cause of the increased morbidity and mortality in patients with insulin dependent diabetes. The prevalence of microalbuminuria was determined in adults with insulin dependent diabetes of five or more years' duration that had started before the age of 41. All eligible patients (n = 982) attending a diabetes clinic were asked to collect a 24 hour urine sample for analysis of albumin excretion by radioimmunoassay; 957 patients complied. Normoalbuminuria was defined as urinary albumin excretion of less than or equal to 30 mg/24 h (n = 562), microalbuminuria as 31-299 mg/24 h (n = 215), and macroalbuminuria as greater than or equal to 300 mg/24 h (n = 180). The prevalence of microalbuminuria and macroalbuminuria was significantly higher in patients whose diabetes had developed before rather than after the age of 20. The prevalence of arterial hypertension increased with increased albuminuria, being 19%, 30%, and 65% in patients with normoalbuminuria, microalbuminuria, and macroalbuminuria respectively. The prevalence of proliferative retinopathy and blindness rose with increasing albuminuria, being 12% and 1.4%, respectively, in patients with normoalbuminuria, 28% and 5.6% in those with microalbuminuria and 58% and 10.6% in those with macroalbuminuria. An abnormal vibratory perception threshold was more common in patients with microalbuminuria (31%) and macroalbuminuria (50%) than in those with normoalbuminuria (21%). This study found a high prevalence (22%) of microalbuminuria, which is predictive of the later development of diabetic nephropathy. Microalbuminuria is also characterised by an increased prevalence of arterial hypertension, proliferative retinopathy, blindness, and peripheral neuropathy. Thus, urinary excretion of albumin should be monitored routinely in patients with insulin dependent diabetes.

The trefoil peptides, a recently recognized family of protease-resistant peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as growth factors, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil factor (RITF and HITF) to subconfluent nontransformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or nontransformed fibroblasts (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6-fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a 15-fold enhancement in restitution. No synergistic effect was observed by the addition of nonmucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGF beta bioactivity, trefoil peptide, and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGF-beta and was not affected by the presence of immunoneutralizing anti-TGF beta antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.

A simple bioassay is described for insulin-like activity (ILA) which is based on the incorporation of (3H)glucose into the lipids of isolated rat fat cells. Total lipids were extracted by adding toluene-based scintillation fluid directly to the incubation flasks, and the radioactivity in the lipids measured by liquid scintillation counting. Insulin dose-response curves with (2-3H)-, (3-3H) and (6-3H)glucose were compared and those obtained with (2-3H)glucose and (3-3H)glucose were found suitable for routine use.

In order to grow in a simple salts medium in which ammonia provides the sole source of nitrogen, micro-organisms must possess some mechanism for the synthesis of amino acids from ammonia and intermediary metabolites. In many bacteria this requirement is fulfilled by the enzyme glutamate dehydrogenase (EC 1.4.1.4), which reductively aminates 2-oxoglutarate to glutamate. In other bacteria (particularly in some species of Bacillus) analogous amino acid dehydrogenases (e.g. alanine dehydrogenase and leucine dehydrogenase) func- tionally replace glutamate dehydrogenase and glutamate is then formed by an aminotransferase reaction.

Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VIIa, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VIIa molecule, namely, 10 gamma-carboxylated, N-terminally located glutamic acid residues, 1 beta-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VIIa as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VIIa. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradations, the protein backbone of recombinant factor VIIa was found to be identical with human factor VIIa. Neither recombinant factor VIIa nor human plasma factor VIIa was found to contain beta-hydroxyaspartic acid. In human plasma factor VIIa, the 10 N-terminally located glutamic acid residues were found to be fully gamma-carboxylated whereas 9 full and 1 partial gamma-carboxylated residues were found in the corresponding positions of the recombinant factor VIIa molecule. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VIIa. In the recombinant factor VIIa, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VIIa and human plasma factor VIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Four peptides present in completely different biological sources have been shown to exhibit a large degree of structural similarity. The peptides include: (i) a 60 amino acid residue breast cancer associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF-7; (ii) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and pancreatic juice; and (iii) a 49 and 50 amino acid residue peptide predicted from a cDNA isolated from the skin of the frog, Xenopus laevis. These peptides are characterized by having one (pS2 and the frog peptides) or two (PSP) domains of a highly conserved 38-39 amino acid residue consensus sequence not found in any other known peptides or proteins. The domain sequences contain 6 cysteine residues in nearly the same positions and it is suggested that these 6 residues are linked by 3 disulphide bonds to form a characteristic 'trefoil' disulphide loop structure common in all four peptides. From the sources of which the peptides have been isolated and from experiments showing that PSP has a growth factor stimulatory effect on MCF-7 cells, it is further suggested that these peptides may represent members of a new family of growth factors.

A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor alpha 1 leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.

We have compared the plasminogen activating capacity of one- and two-chain urokinase-type plasminogen activator (u-PA). In a 125I-plasminogen conversion assay in the presence of high amounts of a plasmin inhibitor, one-chain u-PA pretreated with diisopropyl fluorophosphate had no detectable activity, the detection limit corresponding to the activity of a 400-fold lower amount of two-chain u-PA. In coupled assays in which generated plasmin was measured with a synthetic substrate, activity was clearly observed with the one-chain preparation, but the initial rate of plasminogen activation was lower than that of a 250-fold smaller concentration of two-chain u-PA. The coupled assays for one-chain u-PA are self-activating because plasmin catalyzes conversion of one- to two-chain u-PA, and it is not possible to decide whether the low activity of one-chain u-PA observed with this type of assay is intrinsic or due to contaminations. On the basis of these findings and a discussion of previous studies, it is concluded that one-chain u-PA has a variety of properties similar to the one-chain proenzyme forms of other serine proteases and that it should, therefore, be considered as a genuine proenzyme form of u-PA.

The likelihood ratio test for cointegrating rank is analyzed for partial (or conditional) systems in the vector autoregressive error-correction model. Under the assumption of weak exogeneity for the cointegrating parameters, the asymptotic distributions are given and tables of critical values are provided. A discussion is given of some of the assumptions of the model, why they are needed, and how they are tested.

The plasma C-peptide immunoreactivity (CPR) in 10 normal subjects varied considerably when measured with different antisera in parallel assays. The CPR level correlated with the blank "CPR" value measured in plasma devoid of C-peptide and to a lesser degree with the sensitivity of the standard curves obtained with the individual antisera. Storage of plasma samples at different temperatures and for different lengths of time before the analyses were carried out resulted in further variation in the CPR results. This was caused by a time- and temperature-dependent fall in CPR, which was more pronounced with some antisera than with others. This sensitivity to storage of plasma did not correlate with the antigenic characteristics of the antisera as determined by their reactivity with 11 specific fragments of the C-peptide molecule. The contribution of human proinsulin to the CPR concentration relative in normal subjects was considered to be negligible even though the relative immunoreactivity of human proinsulin and C-peptide ranged from 11 to 143 per cent among these antisera. These results suggest that differences in C-peptide antisera are a major reason for the variation in the concentration of circulating CPR as measured in different C-peptide immunoassays.

Abstract: (R)‐N ‐[4,4‐Bis(3‐methyl‐2‐thienyl)but‐3‐en‐l‐yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of γ‐[ 3 H]aminobutyric acid ([ 3 H]GABA) uptake in a rat forebrain synaptosomal preparation (IC 50 = 67 n M ) and in primary cultures of neurons and astrocytes. Inhibition of [ 3 H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [ 3 H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of [ 3 H]GABA uptake. Binding to benzodiazepine receptors, histamine H 1 receptors, and 5‐hydroxytryptamine lA receptors was inhibited by NO 328 at 5—30 μ M , whereas several other receptors and uptake sites were unaffected. [ 3 H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCI. The specific binding of [ 3 H]NO 328 was inhibited by known inhibitors of [ 3 H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [ 3 H]NO 328 is 18 n M , and the B max is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl‐dependent [ 3 H]NO 328 binding followed that of synaptosomal [ 3 H]GABA uptake. It is concluded that NO 328 is a potent and selective inhibitor of neuronal and glial GABA uptake and that [ 3 H]NO 328 is a useful radioligand for labeling the GABA uptake carrier in brain membranes. In the mouse brain in vivo, [ 3 H]NO 328 likewise showed saturable and reversible binding that could be displaced by analogues of NO 328. Further studies are needed to demonstrate whether the uptake carrier is indeed labeled by [ 3 H]NO 328 in vivo.

A B

The gene encoding subtilisin Carlsberg from Bacillus licheniformis has been isolated by molecular cloning using a mixture of synthetic oligonucleotides. The entire nucleotide sequence of the coding sequence as well as 5' and 3' flanking sequences have been determined. The deduced amino acid sequence reveals an N-terminal signal peptide consisting of 29 residues, a pro-peptide of 76 residues followed by the mature protein comprising 274 residues. The ATG initiator codon is preceded by two putative overlapping ribosomal binding sequences. A palindromic sequence typical for transcription termination is found downstream from the TAA stop codon. Structural comparisons between different known subtilisin genes reveal extensive homology, particularly in the parts coding for the pro-region and the mature protein. Expression studies in Bacillus subtilis show that the cloned fragment produces a functional enzyme when inserted after a B. subtilis promoter.

Neural networks provide a basis for semiempirical studies of pattern matching between the primary and secondary structures of proteins. Networks of the perceptron class have been trained to classify the amino-acid residues into two categories for each of three types of secondary feature: alpha-helix or not, beta-sheet or not, and random coil or not. The explicit prediction for the helices in rhodopsin is compared with both electron microscopy results and those of the Chou-Fasman method. A new measure of homology between proteins is provided by the network approach, which thereby leads to quantification of the differences between the primary structures of proteins.

The trifluoroacetic acid‐mediated removal of t‐butyl groups in protected amino acids leads to the formation of t‐butyl trifluoroacetate. This t‐butyl ester alkylates in trifluoroacetic acid methionine and tryptophan. The t‐butyl trifluoroacetate ester can be destroyed by scavengers commonly employed for t‐butyl cations, and the reaction rates of the scavengers with the ester are used in the evaluation of scavengers. Scavengers of sulphide structure react with t‐butyl trifluoroacetate to form sulphonium compounds, which possess alkylating properties. In the presence of a scavenger during acidolysis, the trifluoroacetic acid and the scavenger will compete in reacting with the t‐butyl cations. Kinetic studies show comparable reaction rates with thiophenol as scavenger. The usefulness of adding scavengers to trifluoroacetic acid in deblocking reactions is due to the removal of t‐butyl trifluoroacetate in addition to the removal of t‐butyl cations. Isobutene reacts with trifluoroacetic acid and yields t‐butyl trifluoroacetate. The reaction reaches an equilibrium displaced in favour of the ester at room temperature. Hence no isobutene can be expected to escape during a deblocking reaction in trifluoroacetic acid.

The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val- Gly-Leu - Met.NH2. This amino acid sequence represents residues (72-92) of gamma-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-gamma, lacks residues (3-17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of beta- and gamma-preprotachykinins in the rabbit gut are different.

A rapid and efficient procedure for site specific mutagenesis is described. A double primed synthesis with a 17-mer mismatch primer and a "universal" 15-mer M13 sequencing primer was used to introduce a T to A transversion into an ompF signal peptide gene cloned in the M13mp8 vector. The two primers were annealed to the circular single stranded M13 template. After a short extension and ligation reaction, a double stranded restriction fragment containing the mismatch (ompF*/EcoR1-SalI) was cut out of the partly single stranded circular DNA and inserted into pBR322. 42% of the E.coli transformants harboured plasmid with the desired mutation, which could be detected by the appearance of a new restriction site (MboII) and by dot blot hybridization of plasmid DNA with the 32P-labeled 17-mer.