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Objective:To assess the tolerability and efficacy of photodynamic therapy (PDT) with RLP068, a novel phthalocyanine-derived photosensitiser, in controlling the bacterial load in different leg ulcers, due to vascular pathophysiology.Method:An observational study of patients with infected leg ulcers of different pathophysiology, admitted to the hospital for a skin grafting procedure, were included. All patients underwent two sessions of PDT at time zero and after 72 hours. A semi-quantitative swab was taken before and 30 minutes after both sessions of PTD. During the time interval between the two treatments the ulcer was covered with foam dressing and compression therapy was applied to all patients. No systemic or topical antibiotics were administered. Statistical analysis of results was performed.Results:A total of 36 patients (13 males, 23 females; aged 72.4±8.6 years, range: 55-85 years) were recruited; two had ulcers on both legs. PDT was shown to be effective in reducing bacterial load after the first treatment. Before the second PDT treatment (72 hours after first PDT treatment), a slight increase of the bacterial load was observed in all ulcers. However, after the second PDT session, bacterial swab results were negative in all but two ulcers. The procedure was well tolerated in all but four patients, who reported a very severe pain at baseline, which increased during treatment.Conclusion:In this study, PDT was effective in reducing bacterial load in patients with infected vascular leg ulcers, and allowed successful skin grafting to take place in all patients. The treatment was generally well tolerated. Studies with a greater number of patients and a control group are planned to confirm these results.

We aimed to evaluate the efficacy of the use of the electromagnetic distal targeting system in treating humeral shaft fracture.

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Background: Glucocorticoids are powerful and effective anti-inflammatory and immunosuppressive drugs. They have extensive use in the treatment of different diseases, even though their side effects, such as hypertension, osteoporosis and, in particular, diabetes, are well known. They can exacerbate hyperglycemia in patients with diabetes mellitus or facilitate the development of metabolic disease in apparently healthy subjects, the so-called steroid-induced diabetes mellitus. The last one is an independent risk factor for other complications associated with the use of glucocorticosteroids. Objectives: We tried to clarify the physiopathology and the correct management of glucocorticoidsinduced diabetes, referring in particular to diabetic patients who have to take such drugs for long periods. Methods: A thorough literature search was performed using Pubmed database to cover all relative reports, using combinations of keywords: diabetes mellitus, insulin, glucocorticoids therapy, dipeptidyl peptidase- 4 inhibitors (DPP-4 inhibitor), glucagon-like peptide-1 receptor agonists (GLP-1 receptor agonists), sodium-glucose co-transporter-2 inhibitors (SGLT-2 inhibitor), hyperglycemia, glucose-lowering drugs. Results & Conclusion: Glucocorticoids-induced diabetes is a harmful and underestimated problem when the therapeutic program is not correct and well planned. Screening or more stringent monitoring must always be suggested before starting with corticosteroids; moreover, the hypoglycemic therapy should be personalized, based on the severity of hyperglycemia, type of steroid used and patients’ comorbidities. All the oral glucose-lowering drugs can be used, but insulin is the most studied drug and the medication recommended for severe hyperglycemia, for patients with pre-existing diabetes or for those patients who have to undergo prolonged therapy over time.

Patients with heart failure (HF) are characterized by relevant problems during sleep, including short sleep time, low sleep quality, and sleep-disordered breathingApproximately 33% of patients with HF have insomnia, potentially related to HF features, adverse effects of medications, or to conditions that often accompany chronic diseases such as mood disorders and psychological stressACC/AHA guidelines have identified sleep deprivation and poor sleep quality as barriers to self-care and treatment adherence in patients with HFSleep-disordered breathing is highly prevalent in patients with HF; both central and obstructive sleep apnoeas are frequently observed in these patients, and were shown to have an important added prognostic valueContinuous positive airway pressure has a beneficial effect on left ventricular ejection fraction and is currently the best treatment option for obstructive sleep apnoeas in patients with HFAt present, no consensus exists on whether central sleep apnoeas should be treated and what the optimal therapy in HF might be

On the occasion of the 140th anniversary of the birth of the famous Italian mathematician Beppo Levi (1875–1961), we publish an interview with his daughter Emilia. We also recall his brother Eugenio Elia Levi, a well-known and brilliant mathematician whose life was cut short at the front during World War I. A brief outline of Beppo Levi’s life is followed by an introduction to a letter that he wrote to the periodical Israel in 1919, in part regarding the foundation of the state of Israel. Finally, we publish an English translation of the letter in its entirety.

Objective:The aim of the study was to compare the efficacy of a microorganism-binding (MB) dressing with a silver-containing hydrofiber (SCH) dressing in controlling the bacterial loads of heavily colonised or locally infected chronic venous leg ulcers, before surgical management with homologous skin grafts.Method:A randomised comparative single centre study recruited patients presenting with hard-to-heal critically colonised or locally infected leg ulcers, who could be treated with skin grafting. Inclusion criteria included; ulcers of vascular aetiology, over 18 years old, a wound duration ≥6 months and ankle brachial index (ABPI) >0.6. Patients were randomly assigned to treatment with SCH dressings (Aquacel Ag) or MB dressing (Cutimed Sorbact). Dressings were changed daily over a four-day observation period, after which they were taken for a skin grafting procedure. Swab samples from ulcer beds were taken in order to quantify the bacterial load at inclusion (D0) and at the end of the observation period day 4 (D4). No antibiotics were administered before or during the evaluation period.Results:Both groups (n=20 SCH, n=20 MB) were similar in gender, age, pathophysiology (both had 15 patients with venous leg ulcers and 5 with arterial leg ulcers), ulcer surface, ulcer duration, treatment-related pain and initial bacterial load. Analysing bacterial load variation showed a significant reduction of bacterial burden at D4 in both groups. In the SCH group, we found an average bacterial load reduction of 41.6%, with an average reduction of 73.1% in the MB group (p< 0.00001). No serious adverse events were reported.Conclusion:Our evaluation confirmed that MB and SCH dressings are effective in reducing the bacterial burden in critically colonised or locally infected chronic leg ulcers, without inducing adverse events, with MB dressings significantly more effective.Declaration of interest:There were no external sources of funding for this study. The authors have no conflicts of interest to declare.

Abstract: Background:: The incidence of Crohn’s disease (CD) has been shown to be lower in Southern than in Northern Europe. Data on the frequency of the NOD2/CARD15 mutations for Mediterranean area are very scant. Aim:: To determine the incidence of CD from 1979 to 2002 in a township in Sicily together with the allele frequency of NOD2/CARD15 mutations in patients, family members and controls, and to determine the allele frequency of these mutations in sporadic CD from other areas of Sicily in comparison with a control population. Methods:: Casteltermini is a small town close to Agrigento (Sicily) with a population of 9,130 inhabitants. All the diagnoses of inflammatory bowel disease (IBD) made from 1979 to 2002 were obtained through the local health authority. NOD2/CARD15 mutations were studied in 23 out of the 29 patients with CD in Casteltermini, in 60 family members and in 64 controls. NOD2/CARD15 was also studied in 80 sporadic cases of CD disease among Sicilians outside Casteltermini and 118 healthy controls. Results:: From 1979 to 2002, 29 patients with CD and 13 patients with ulcerative colitis (UC) were registered. The 6-year mean incidence of CD ranged from 8.0 to 17 new cases for every 100,000 inhabitants, whereas the mean incidence of UC ranged from five new cases to 7.8 for every 100,000 inhabitants. The allele frequencies of NOD2/CARD15 mutations (L1007finsC, G908R, R702W) were 8.7, 4.3 and 8.7%, respectively, in CD cases; 5.0, 4.2 and 3.1% in family members; 1.6, 2.3 and 3.1% in controls. In sporadic Sicilian CD patients outside Casteltermini the allele frequency was 7.5, 8.1, 6.2% whereas in control population it was 3.3, 1.6, 1.6%. Conclusions:: A high incidence of CD compared with UC was observed in this small town in Southern Italy. The frequency of NOD2/CARD15 mutations in CD is similar to other Caucasian population studied so far.

To enhance the delivery of rhGAA (recombinant GAA, where GAA stands for acid α-glucosidase) to the affected muscles in Pompe disease, the carbohydrate moieties on the enzyme were remodelled to exhibit a high affinity ligand for the CI-MPR (cation-independent M6P receptor, where M6P stands for mannose 6-phosphate). This was achieved by chemically conjugating on to rhGAA, a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the receptor. The carbonyl chemistry used resulted in the conjugation of approx. six synthetic ligands on to each enzyme. The resulting modified enzyme [neo-rhGAA (modified recombinant human GAA harbouring synthetic oligosaccharide ligands)] displayed near-normal specific activity and significantly increased affinity for the CI-MPR. However, binding to the mannose receptor was unaffected despite the introduction of additional mannose residues in neo-rhGAA. Uptake studies using L6 myoblasts showed neo-rhGAA was internalized approx. 20-fold more efficiently than the unmodified enzyme. Administration of neo-rhGAA into Pompe mice also resulted in greater clearance of glycogen from all the affected muscles when compared with the unmodified rhGAA. Comparable reductions in tissue glycogen levels in the Pompe mice were realized using an approx. 8-fold lower dose of neo-rhGAA in the heart and diaphragm and an approx. 4-fold lower dose in the skeletal muscles. Treatment of older Pompe mice, which are more refractory to enzyme therapy, with 40 mg/kg neo-rhGAA resulted in near-complete clearance of glycogen from all the affected muscles as opposed to only partial correction with the unmodified rhGAA. These results demonstrate that remodelling the carbohydrate of rhGAA to improve its affinity for the CI-MPR represents a feasible approach to enhance the efficacy of enzyme replacement therapy for Pompe disease.

Posttranslational modification can influence the biologic activity of recombinant proteins. The effects of β-subunit C-terminal truncation, oligosaccharide heterogeneity, and chemical oxidation on the in vitroactivity of recombinant human thyroid-stimulating hormone (rhTSH) were investigated. β-Subunit C-terminal truncation up to residue 113 did not effect the in vitroactivity of the hormone. The relationship between the heterogeneity of oligosaccharide structures on rhTSH and specific activity of the glycoprotein hormone was also examined. Oligosaccharide profiles were generated for preparations of rhTSH containing similar sialic acid levels. A weak correlation was observed between relative levels of monosialylated biantennary, bisialylated biantennary, and trisialylated triantennary oligosaccharide species and in vitroactivity of the recombinant hormone (p < 0.05).To examine the effect of chemically induced methionine oxidation on the activity of rhTSH, the hormone was treated with tert-butyl hydroperoxide and then characterized. Using peptide mapping and mass spectrometry, the degree of oxidation of the five methionine residues within rhTSH was measured. Met-71 in the α-subunit was the most susceptible to oxidation whereas Met-9 in the β-subunit was the most resistant. Also, after tert-butyl hydroperoxide treatment, levels of oxidation of Met-32 in the β-subunit, and Met-29 and Met-47 in the α-subunit were less than half of that observed for Met-71. The in vitroactivity of rhTSH initially declined with increasing oxidation; however, the loss in activity plateaued at approximately 50% of the control sample activity. In summary, despite the possible effects that posttranslational modifications may have on the bioactivity of a protein, a limited degree of variation in bioactivity was observed for the rhTSH preparations described in this study.

A cDNA of the human terminal deoxynucleotidyltransferase (TdT; "terminal transferase," EC 2.7.7.31) was isolated from a human lymphoblastoid cell cDNA library in lambda gt 11 by using immunological procedures. Four inserts containing 723 to 939 base pairs were recloned in pBR322 for hybridization and preliminary sequence studies. mRNA selected by hybridization to recombinant DNA was translated to a 58-kDa peptide that specifically immunoprecipitated with rabbit antibodies to calf terminal transferase and mouse monoclonal antibody to human terminal transferase. Blot hybridization of total poly(A)+ RNA from KM3 (TdT+) cells with nick-translated pBR322 recombinant DNA detected a message of about 2000 nucleotides, sufficient to code for the 580 amino acids in the protein. mRNA from terminal transferase- cells gave no signal in hybrid selection or RNA blot hybridization. The complete sequence of the 939-base-pair insert sequence was obtained from deletions cloned in pUC8. The DNA sequence contains an open reading frame coding for 238 amino acids, about 40% of the protein. Three peptides isolated by HPLC from tryptic digests of succinylated 58-kDa calf thymus terminal transferase were sequenced, providing 20, 18, and 22 residues of peptide sequence. A search of the translated sequence of the 939-base-pair insert shows three regions beginning after arginine that have greater than 90% homology with the sequence determined from the calf thymus terminal transferase peptides. These results provide unambiguous evidence that the human terminal transferase sequence has been cloned.

Theory X leadership has helped to bring ed ucation to the brink of disaster, says this writer, who claims it is time for Theory Y.

We have purified two 35-kDa proteins from rat peritoneal lavages that inhibit phospholipase A2 activity. Both are calcium/phospholipid-dependent membrane binding proteins and share similar structural and biochemical properties with lipocortins I and II. By sequence analysis we confirmed that they are lipocortin-related, and we refer to the two inhibitors as lipocortins III and V. Using partial sequence information obtained from the purified rat proteins, full length cDNA clones for both proteins and for their human counterparts were isolated. As with lipocortins I and II, the amino acid sequences of lipocortins III and V which were deduced from the cDNA clones are highly conserved, sharing 50% identity with other family members. Related proteins were also purified from bovine intestinal mucosa and characterized by peptide mapping, sequence, and immunological analyses. In addition to lipocortins III and V the bovine preparation contained a third 35-kDa inhibitor and a 68-kDa inhibitor, extending the number of known lipocortins to six distinct proteins. While the various lipocortins are structurally similar, distinct differences in their cellular distribution indicate specialized roles for the individual proteins.

Mullerian inhibiting substance (MIS) is a differentiation factor that causes the Mullerian duct to regress during the development of the male reproductive tract. The active form is a disulfide-linked dimer consisting of two identical 70-kDa subunits. Recently, the amino acid sequence for MIS was deduced from its gene sequence and revealed that the carboxyl-terminal region shares homology with transforming growth factor (TGF)-beta. Since TGF-beta is produced as a large latent precursor that requires proteolytic activation for activity, we sought to determine if MIS might undergo a similar processing event. Here we demonstrate that typically 5 to 20% of the protein in MIS preparations is cleaved at a site 109 amino acids from the carboxyl terminus. Concurrent cleavages from both chains of the MIS dimer produces a 25-kDa TGF-beta-like fragment and a high molecular mass complex derived from the amino terminus of the protein. Although the two fragments are noncovalently linked, they remain tightly associated after cleavage, and thus are structurally organized like TGF-beta within its precursor. The same cleavage products also can be generated by limited proteolysis with plasmin, which provides a simple method for converting the entire preparation into the cleaved form. The plasmin-digested MIS is fully active in the organ culture assay.

Two homologous proteins, murine (m) and human (h) granulocyte–macrophage colony–stimulating–factor (GM–CSF), were differentially glycosylated and processed during secretion by a yeast host. Secreted hGM–CSF, but not mGM–CSF was significantly O–glycosylated. Human GM–CSF was expressed mainly as a highly N–glycosylated form, while 50% of mGM–CSF was unglycosylated, or core–glycosylated. The Asn–X–Thr and Asn–X–Ser sequences of hGM–CSF were equally efficient sites for N–glycosylation, which enhanced the secretion efficiency of hGM–CSF, but not of mGM–CSF. N–terminal analysis of secreted hGM–CSF revealed a novel processing site following the pre–region of the MFα1/hGM–CSF precursor and in addition, suggested partial N–terminal degradation by the STE13–encoded dipeptidase. GM–CSFs secreted by yeast and by animal cells had identical in vitro biological activities.

Polyneuritis is a rare neurological complication of typhoid fever. A clinical and electrophysiological investigation of a very severe and purely motor form of typhoid polyneuritis is reported.

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2and PI(3,4,5)P3as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.

A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.

We have deduced the 1675-amino acid sequence of rat clathrin heavy chain from cDNA clones and predict a protein of Mr 191,569. We have established the polarity of the heavy chain and assigned sequence positions to several structural landmarks of the clathrin leg. The terminal domain at the distal end of the clathrin leg is at the amino terminus of the heavy chain. It is connected to the distal segment by a flexible "link" from Tyr-479 to Arg-523. There is an unusual sequence at the carboxyl terminus that may form the globular projection at the vertex of the clathrin trimer. We suggest that a possible site of heavy-chain-light-chain interaction is located in the proximal segment. Comparison with other partially sequenced mammalian clathrin heavy chains shows that the primary structure is highly conserved. The heavy chain is unrelated to other classes of structural proteins.

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.

5-Ketofructose reductase [D(-)fructose:(NADP+) 5-oxidoreductase] was purified to homogeneity from Erwinia citreus and demonstrated to catalyse the reversible NADPH-dependent reduction of 5-ketofructose (D-threo-2,5-hexodiulose) to D-fructose. The enzyme appeared as a single species upon analyses by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing with an apparent relative molecular mass of 40,000 and an isoelectric point of 4.4. The amino acid composition of the enzyme and the N-terminal sequence of the first 39 residues are described. The steady-state kinetic mechanism was an ordered one with NADPH binding first to the enzyme and then to 5-ketofructose, and the order of product release was D-fructose followed by NADP+. The reversible nature of the reaction offers the possibility of using this enzyme for the determination of D-fructose.

The genes that encode the α, β, and γ subunits of component C of methyl coenzyme M reductase (mcrA, mcrB, and mcrG) in Methanococcus vannieliihave been cloned and sequenced, and their expression in Escherichia colihas been demonstrated. These genes are organized into a five-gene cluster, mcrBDCGA, which contains two genes, designated mcrCand mcrD, with unknown functions. The mcrgenes are separated by very short intergenic regions that contain multiple translation stop codons and strong ribosomebinding sequences. Although the genome of M. vannieliiis 69 mol% A+T, there is a very strong preference in the mcrA, mcrB, and mcrGgenes for the codon with a C in the wobble position in the codon pairs AAUC(asparagine), GAUC(aspartic acid), CAUC(histidine), AUUC(isoleucine), UUUC(phenylalanine), and UAUC(tyrosine). The mcrCand mcrDgenes do not show this codon preference and frequently have U or A in the wobble position. As the codon pairs listed above are likely to be translated by the same tRNA with a G in the first anticodon position, the presence of C in the wobble position might ensure maximum efficiency of translation of transcripts of these very highly expressed genes.

To examine the envelope proteins of duck hepatitis B virus (DHBV), which are encoded by the pre-S/S open reading frame of the viral genome, an antiserum was raised in rabbits against a fusion protein comprising most of the pre-S coding segment. By using this antiserum, viral particles could be precipitated from serum, and two pre-S proteins with molecular sizes of approximately 35 and 37 kilodaltons were detected in the sera and livers of DHBV-infected ducks after Western blotting and after biosynthetic labeling of a primary duck liver cell culture. In serum, the pre-S proteins were shown to exist predominantly in DHBV-DNA-free particles associated with a 17-kilodalton protein which, by N-terminal amino acid sequence analysis, was shown to represent the viral S protein which is encoded by the 3' proximal segment of the DHBV pre-S/S open reading frame. To compare the immunogenic potential of the S and pre-S proteins, serum particles and gel-purified S protein were used to immunize rabbits. In neither case was a significant immune response against the DHBV S protein observed. However, a good antibody titer against DHBV pre-S was obtained even after immunization with small amounts of the pre-S antigen.

We have purified two low-molecular-weight polypeptides from the Prague C strain of Rous sarcoma virus and have identified these as products of the gag precursor Pr76 by protein sequencing and by amino acid analysis. Both polypeptides are derived from a stretch of 22 amino acids within Pr76 that separates p19 and p10. We refer to this region as p2. Together the two cleavage products form the entire p2 region. The junctions of p19 with the amino-terminal fragment of p2 and of p10 with the carboxy-terminal fragment of p2 define two new processing sites within the gag precursor, Tyr-155-His-156 and Gly-177-Ser-178. Both polypeptides are major cleavage products of Pr76 that occur in Prague C Rous sarcoma virus at an estimated 1,000 copies per virion. They also are prominent components of avian myeloblastosis virus. The combination of gel filtration and reverse-phase high-pressure liquid chromatography, which was used for the isolation of the two fragments of p2, resolved over a dozen other low-molecular-weight polypeptides from avian sarcoma and leukemia viruses that previously were undetected. This technique thus should serve as a useful procedure for further characterization of viral components.

We report the production, purification, characterization, and partial amino acid sequence of a plasminogen inhibitor (PA-I). The starting material is culture fluid from phorbol myristate 13-acetate-treated U-937 cells and the isolation steps consist of preparative isoelectric focusing followed by affinity chromatography on Cibacron Blue-Sepharose. PA-I migrates as a closely spaced doublet of 47-kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forms covalent complexes with urokinase and two-chain tissue-type plasminogen activator, displaying second order rate constants of 0.9 X 10(6) M-1 s-1 and 0.2 X 10(6) M-1 s-1, respectively. Upon treatment with 1 M NH4OH, the covalent complexes were hydrolyzed, yielding a 35-kDa inhibitor fragment. A partial amino acid sequence of PA-I showed that it belongs to the antithrombin III family of inhibitors. PA-I is immunologically related to a PA-inhibitor from human placenta. mRNA from phorbol myristate 13-acetate-treated U-937 cells directed, in a rabbit reticulocyte derived cell-free system, the biosynthesis of only one 47-kDa protein that could be immunoprecipitated with anti-PA-I IgG, indicating that the two molecular forms of PA-I are the products of post-translational processing.

We have isolated the cDNA for human lymphocyte function-associated antigen 3 (LFA-3), the ligand of the T lymphocyte CD2 molecule. The identity of the clones was established by comparison of the deduced amino acid sequence to the LFA-3 NH2-terminal and tryptic peptide sequences. The cDNA defines a mature protein of 222 amino acids that structurally resembles typical membrane-anchored proteins. An extracellular domain with six N-linked glycosylation sites is followed by a hydrophobic putative transmembrane region and a short cytoplasmic domain. The mature glycoprotein is estimated to be 44-68% carbohydrate. Southern blots of human genomic DNA indicate that only one gene codes for human LFA-3. Northern blot analysis demonstrates that the LFA-3 mRNA of 1.3 kb is widely distributed in human tissues and cell lines.

Human lipocortin I is a 38.5-kDa phospholipase A2 inhibitor that has been produced in Escherichia coli in large quantities by recombinant DNA technology (Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, E.P., Browning, J.L., Ramachandran, K.L., and Pepinsky, R.B. (1986) Nature 320, 77-80). To localize the region within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis with elastase and characterized their structure by sequencing and peptide mapping. Five active fragments have been analyzed in detail. The smallest is an 18-kDa fragment derived from the amino-terminal half of lipocortin. Three of the larger fragments contain this region. The fifth fragment is missing 83 amino acids from the amino terminus. A region common to all the active fragments (amino acid residues 97-178) is 70% homologous with the corresponding region from a second member of the lipocortin family which recently was cloned (Huang, K-S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B. (1986) Cell 46, 191-199) and thus presumably is important for activity. In addition to inhibitory fragments, we have isolated a 3-kDa proteolytic fragment from the amino terminus of lipocortin I that contains the known phosphorylation site for protein-tyrosine kinases. Because of sequence homology of the 3-kDa fragment with biologically active synthetic peptides from pp60v-src and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin.

The skins of obese Zucker rats contained per g wet weight significantly more lipid and significantly less water than did the skins from age and sex matched lean Zucker rats. The obese skins also contained less collagen than the lean skins when compared on the basis of mg collagen per g wet weight, per g dry weight, or per cm2of skin; however, no difference was found when the skins were compared on the basis of mg collagen per g lipid-free dry weight. The solubility properties of the collagen from obese and lean skins were the same while the break strength of the obese skins was decreased. The results indicate that skins from obese Zucker rats have a normal collagen composition but an abnormal lipid composition. The reduced break strength of the skin from obese rats, due probably to the decreased amount of collagen per cm2of skin caused by a displacement of collagen fibers by lipid, may explain the frequent skin tearing observed in the obese rats.

The anti-inflammatory action of glucocorticoids has been attributed to the induction of a group of phospholipase A2inhibitory proteins, collectively called lipocortin. These proteins are thought to control the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, by inhibiting the release of their common precursor, arachidonic acid, a process that requires phospholipase A2hydrolysis of phospholipids1–11. Lipocortin-like proteins have been isolated from various cell types, including monocytes3, neutrophils4and renal medullary9cell preparations. The predominant active form is a protein with an apparent relative molecular mass (Mr) of 40,000 (40K). These partially purified preparations of lipocortin mimic the effect of steroids, and mediate anti-inflammatory activity in various in vivo model systems3,5,11. Using amino-acid sequence information obtained from purified rat lipocortin, we have now cloned human lipocortin complementary DNA and expressed the gene in Escherichia coli. Our studies confirm that lipocortin is a potent inhibitor of phospholipase A2activity.

Clonazepam (1 mg/kg) and antipyrine (0.1 mg/kg) were administered simultaneously by intravenous bolus injection to three dogs. After 2 weeks of chronic phenobarbital administration, the studies were repeated. Plasma concentration-time curves in all studies were biexponential. Phenobarbital administration increased total plasma clearances of clonazepam and antipyrine by 102% and 98.5%, respectively. Volumes of distribution were not altered, and consequently reductions in terminal exponential half-lives observed after phenobarbital were attributed to increases in clearances.

Isoniazid was administered orally to two slow acetylator subjects, and plasma and saliva samples were assayed for intact drug. Saliva/plasma concentration ratios tended to be scattered about the value of 1. The results indicate that salivary level measurements may provide a useful approach for acetylation phenotyping. However, because of instability of isoniazid in frozen specimens, analyses of drug levels had to be initiated within 1 hr from the time of collection. Protein binding studies indicated that INH is not bound to plasma proteins to any appreciable extent.

We have purified from rat peritoneal exudates a 37-kDa protein that inhibits phospholipase A2 activity. It is the predominant phospholipase inhibitor protein in these preparations and also is detected in a wide variety of cell lines. Levels of expression range from 0 to 0.5% of total protein. In the peritoneal preparations, the inhibitor is partially proteolyzed into a series of lower mass forms, including species at 30, 24, and 15 kDa. These fragments all are immunoreactive with an antibody raised against the 37-kDa protein. The rat protein also is immunoreactive with an antibody developed against a 6-kDa phospholipase inhibitor protein from snake venom. The primary structure of more than half of the rat inhibitor has been deduced by protein microsequence analysis. These sequences are closely related to sequences from its human analogue, which we recently cloned and expressed (Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L. K., Foeller, C., Chow, E. P., Browning, J. L., Ramachandran, K. L., and Pepinsky, R. B. (1986) Nature, in press), and thus we infer that the inhibitor is highly conserved evolutionarily. Properties of the molecule suggest that it is a member of a family of steroid-induced anti-inflammatory proteins collectively referred to as lipocortin.

Determining objectives for a school's programs is an important phase of the planning required in PPBS. Here the author stresses the role of the principal in their development and writing as well as his relationship to others in the organization.

The manner in which a membrane protein is anchored to the lipid bilayer may have a profound influence on its function. Most cell surface membrane proteins are anchored by a membrane-spanning segment(s) of the polypeptide chain, but another type of anchor has been described for several proteins: a phosphatidyl inositol glycan moiety, attached to the protein C terminus1,2. This type of linkage has been identified on membrane proteins involved in adhesion3and transmembrane signalling4,5and could be important in the execution of these functions. We report here that an immunologically important adhesion glycoprotein, lymphocyte function-associated antigen 3 (LFA-3), can be anchored to the membrane by both types of mechanism. These two distinct cell-surface forms of LFA-3 are derived from different biosynthetic precursors. The existence of a phosphatidyl-inositol-linked and a transmembrane anchored form of LFA-3 has important implications for adhesion and transmembrane signalling by LFA-3.

The article discusses the evolution of disability management in the United States. Small businesses can collaborate with the disability manager in order to benefit from the expansion of programs and practices. The return-to-work program also addresses non-occupational illnesses and injuries instead of just workers' compensation cases.

The nucleotide sequence of a 1467 bp fragment of Streptomyces hygroscopicus DNA containing the gene (hyg) encoding a hygromycin B phosphotransferase (HPH) has been determined. The N-ter-minal atnino acid sequence of HPH determined by automated Edman degradation has allowed the coding sequence of the hyg gene to be identified. The translation initiation triplet is GTG and 5 bp preceding it there is a sequence complementary to the 3′-end of 16S rRNA from S. lividans. The transcriptional start and termination sites have been determined; the presumptive promoter region has only partial homology to that of the Streptomyces vinaceus vph gene and is different to the promoter sequences of other Streptomyces genes.