Recombinant activated protein C usage in Scotland: a comparison with published guidelines and a survey of attitudes.

Severe sepsis is a common cause of admission to the intensive care unit and is associated with a high hospital mortality. This audit explored the current use of, and attitudes towards, recombinant activated protein C therapy across Scotland, and compared these with current guidance. Patients with severe sepsis were followed for three days. Consideration and/or usage of recombinant activated protein C were compared with two different guidelines. Ninety-seven patients were admitted to the intensive care unit over the audit period. Recombinant activated protein C was used in nine of these patients. Depending on the criteria used, between 50% and 81% of the patients who qualified for recombinant activated protein C therapy did not receive it. Subsequent to the audit, a survey was performed to study intensive care unit consultants' attitudes to recombinant activated protein C therapy. A total of 125 consultants responded to the survey (77%). Of these, 104 (83%) stated that they used recombinant activated protein C in their clinical practice, 56 (52%) of whom prescribed it to patients with two-organ failures and an Acute Physiology and Chronic Health Evaluation II score of ≥ 25. Thirty-nine respondents (38%) stated that two-organ failures alone would be an adequate trigger for therapy. We conclude that recombinant activated protein C is potentially under-used to treat severe sepsis. Many consultants seem to reserve the drug for the most severely ill sub group of patients.

Value of multilevel sectioning for improved detection of micrometastases in sentinel lymph nodes in invasive squamous cell carcinoma of the vulva.

UNLABELLED: Clinical usefulness of sentinel lymph node (SLN) biopsy has been demonstrated in the management of early vulvar cancer. However, what constitutes a negative SLN has not been well defined. Furthermore, to what extent the SLNs should be sectioned for the greatest likelihood of detection of micrometastases and whether multilevel sectioning will further increase this detection rate in this setting have not been well studied. We analyzed 280 groin lymph nodes (SLNs=45, non-sentinel [NSLNs]=235) in 14 patients with invasive squamous cell carcinoma (ISCC) of the vulva treated with vulvectomy and inguinal SLN and NSLN dissection at the H. Lee Moffitt Cancer Center (HLMCC) between 1996 and 2001. Each SNL was evaluated for micrometastases by H&E and pancytokeratin AE1/3 (CKAE1/3) immunohistochemical staining. All negative SNLs (N=40) were sectioned times 3 (x3) at 50-micron intervals and independently reviewed by two pathologists in order to assess the utility of this inexpensive and logical approach to identifying additional micrometastases. Also, the Wilcoxon Rank Sum Test was used to determine if there was an association between tumor size, depth of invasion and SNL status. The patient age ranged from 35 to 81 years (mean 59 yrs); size of invasive tumor from 1.0 to 7.0 cm (mean 3.4 cm); depth of invasion from 3 to 25 mm (mean 10.8 mm). Of 45 SLNs examined from 14 patients, 11% (5/45) SNLs were positive for micrometastases on initial H&E and/or CKAE1/3 stains. Eighty-nine per cent (40/45) SNLs were negative in the remaining 9 patients. None of the latter 40 SNLs showed micrometastases on additional multilevel sectioning. Instead 3 of 135 NSLNs examined in these 9 patients revealed micrometastases on H&E (skip-micrometastases). Mean tumor size (cm) and depth of invasion (cm) were 4.06 (s.d. 1.89) and 1.20 (s.d. 0.35) for SLN (+) and 3.02 (s.d. 2.12) and 1.01 (s.d. 0.86) for SLN (-) tumor subsets (p values 0.385 and 0.348, respectively). CONCLUSION: Following routine H&E and CK AE1/3 stains, multilevel sectioning does not appear to detect additional micrometastases in sentinel lymph nodes in squamous cell carcinoma of the vulva. Even though mean tumor size and depth of invasion were greater in SNL (+) as compared to SLN (-) tumor subsets in our series, this difference did not reach statistical significance.

Recognition of the acceptor beta-D-GlcpNAc-(1-->2)-alpha-D-Manp- (1-->6)-beta-D-Glcp-OR by N-acetyl-glucosaminyltransferase-V: none of the hydroxyl groups on the Glc-residue are important.

The enzyme, N-acetylglucosaminyltransferase-V (GlcNAcT-V, E.C. 2.4.1.155), transfer a beta-D-GlcpNAc residue, from UDP-GlcNAc, to the OH-6 group of the Man residue in the synthetic acceptor beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp-O(CH2)7 CH3 (3). Trisaccharide 3 is an excellent substrate for the enzyme from hamster kidney with a Km value of 26 microM. In this paper we examine the contribution of the Glc residue in 3 to acceptor recognition by this enzyme. beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-O(CH2)7CH3 where the Glc residue in 3 has been deleted, was synthesized and found to be a very poor substrate with a Km value elevated to almost 2 mM. Two other analogues of 3, where the Glc residue was O-trimethylated (6) or O-tribenzylated (7), respectively, possessed Km values very near to those of 3. The Glc residue in 3 is thereby shown to present an important recognition element for GlcNAcT-V, but none of the free hydroxyl groups are required. This observation should facilitate the design of more hydrophobic and membrane-permeable analogues of 3 that are expected to function as specific glycosylation inhibitors.

Acceptor-substrate recognition by N-acetylglucosaminyltransferase-V: critical role of the 4"-hydroxyl group in beta-D-GlcpNAc-(1-->2)-alpha-D-Manp(1-->6)-beta-D-Glcp-OR.

The enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) transfers GlcNAc from UDP-GlcNAc to the OH-6' group of oligosaccharides terminating in the sequence beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp (or Manp)-OR (5, R = (CH2)7CH3) to yield the sequence beta-D-GlcpNAc-(1-->2)-[beta-D-GlcpNAc-(1-->6)]-alpha-D-Manp-(1--> 6)- beta-D-Glcp (or Manp)-OR. Biosynthetically, if beta-(1-->4)-galactosyltransferase acts first on 5, the product beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-be ta-D-Glcp (or Manp)-OR (7) is no longer a substrate for GlcNAcT-V even though it retains the active OH-6' group. The reason for this loss in activity is examined in this paper. Six analogues of the acceptor trisaccharide 5, all with the reducing-end D-gluco configuration, were chemically synthesized. A key feature of the synthetic scheme is the use of 1,2-diaminoethane for the efficient removal of N-phthalimdo protecting groups. In these analogues OH-4 of the terminal sugar unit, the site of galactosylation by GalT in the normal GlcNAc-terminating trisaccharide 5, was systematically replaced by OMe, F, NH2, NHAc, and H, as well as inverted to the galacto configuration. The interactions of the resulting trisaccharide analogues with GlcNAcT-V from hamster kidney were then evaluated kinetically. All six compounds were found to be essentially inactive either as acceptors or as inhibitors of GlcNAcT-V. The conclusion is reached that galactosylation of natural acceptors for GlcNAcT-V destroys acceptor activity, not by introduction of the steric bulk of an added sugar residue, but by destroying an important hydrogen-bonding interaction of terminal OH-4 of the GlcNAc residues with the enzyme. This OH-4 group is therefore designated as a key polar group for GlcNAcT-V.

A plant fucosyltransferase with human Lewis blood-group specificity.

A fucosyltransferase from mung bean seedling was found to transfer L-fucose from GDP-fucose to the Type 1 disaccharide beta-D-Galp-(1----3)-beta-D-GlcpNAc-OR [R = (CH2)8COOMe]. The product, which was detected by an anti-Lea antibody in a novel ELISA assay, was isolated and shown to be the human Lea blood-group determinant beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-OR by 1H-n.m.r. spectroscopy. This enzyme activity is distinct from that of the human Lewis-fucosyl-transferase since alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-beta-D-Glcp-OR is a very poor substrate, while the Type 2 disaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-OR is not an acceptor. In common with the Lewis fucosyltransferase, the H-Type 1 trisaccharide alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-OR was an excellent substrate for the enzyme. This new enzyme activity was further characterized with respect to pH, nucleotide, Mn2+ dependence, and acceptor specificity against a panel of synthetic oligosaccharides.

Mutual visual regard during mother-infant play.

Mutual visual regard was observed in 48 mother-infant dyads during a 6-min. play session. Infant-mother dyads containing 4 mo. -olds displayed significantly more mutual visual regard than dyads containing 6- or 8-mo.-olds. In addition, the more time infants spent in face-to-face interaction with mother, the more smiling they engaged in. No sex difference were observed.

Manic-depressiveness and its correlates.

Manic-depressiveness is the name here given to a hypothesized personality continuum that has, at one extreme, manic-depressive psychosis. A Manic-Depressiveness Scale is described, which comprises three scales, Manic Experience, Depressive Experience, and the sum of the two, since they are correlated. 250 undergraduate psychology students at the University of Adelaide and at Goldsmiths' College, London, were administered the Manic-Depressiveness Scale along with 12 measures including the Eysenck Personality Questionnaire (Revised). Scores on the total Manic-Depressiveness Scale tended (in order of size of association) to be correlated with Schizotypal Personality (and three subscales), Neuroticism, Magical Ideation, Mystical Experience, Belief in the Paranormal, absence of Social Naïveté, and Psychoticism. Manic Experience showed a pattern of relationships with the above variables broadly similar to that of Depressive Experience but included Creative Personality, while Depressive Experience included introversion. The relationship between manic-depressiveness and schizotypy is discussed.

Mother-child interactions involving two-year-olds with Down syndrome: a look at individual differences.

Individual differences in mother-child interaction patterns involving 18 2-year-olds with Down syndrome were examined. Ratings of maternal, child, and dyadic qualities observed in semistructured free play interactions were intercorrelated, and also correlated with Bayley MDI scores. Results indicated that social initiative, social responsivity, and play maturity of the children were positively intercorrelated and significantly correlated with MDI. Maternal sensitivity, elaborativeness, stimulation value, and mood were positively intercorrelated, with stimulation value being the major maternal quality found to be positively correlated with child MDI. A dyadic rating of mutuality was also positively correlated with child MDI. Of special interest was the finding that maternal directiveness and sensitivity were separable dimensions of maternal style, with directiveness as an isolated dimension found to be unrelated to MDI or to other maternal qualities. Exploratory analyses of subgroups of mothers, however, suggested that directiveness and sensitivity interrelated in a number of different patterns.

When did Mrs Thatcher resign? The effects of ageing on the dating of public events.

Two experiments investigated whether, in light of the commonly reported phenomenon of subjective time acceleration with age, there would be an effect of age on the dating of public events. In the first experiment covering the past seven years there was the suggestion of a decrease in forward telescoping with age, and in the second covering the period from 1977-89 this trend was continued, with the over-60s group now showing a tendency to date events too distantly. This effect is uncommon in dating studies and may offer evidence for the existence of time acceleration. An additional finding was that adults in the age range 35-50 years showed greater accuracy in dating events than did university students and adults over the age of 60.

An enzyme-linked immunosorbent assay for N-acetylglucosaminyltransferase-V.

The development of an enzyme-linked immunosorbent assay (ELISA) for uridine 5'-diphospho-N-acetyl-glucosamine: alpha mannoside beta 1----6 N-acetylglucosaminyltransferase (GnT-V) is reported. The assay quantitates the enzymatic conversion of the specific synthetic GnT-V acceptor GlcNAc beta 1----2Man alpha 1----6Man beta-R (5) to the product GlcNAc beta 1----2[GlcNAc-beta 1----6]Man alpha 1----6Man beta-R (6) when these oligosaccharide structures were covalently attached to bovine serum albumin which was then coated on microtiter wells. Conversion of 5 to 6 was detected using a polyclonal antiserum raised against the product 6 and from which antibodies cross-reacting with acceptor 5 had been removed by affinity adsorption. GnT-V activity detected by ELISA was linearly proportional to both enzyme concentration and time under appropriate experimental conditions where 50-300 fmol of product was formed per microtiter well. GnT-V activity could be measured by ELISA in Triton X-100 extracts of hamster kidney acetone powder and in human serum. The twofold increase in GnT-V activity which is known to accompany Rous sarcoma virus transformation of baby hamster kidney cells could also be quantitated using the ELISA.

Maternal language to prelinguistic infants: syntactic aspects.

Maternal speech to children has been shown to vary by age and language ability of the children. Previous studies have usually involved children over 1 year of age. In this study maternal speech to male and female 4-, 6-, and 8-month-old infants was recorded in the laboratory. Mothers used shorter utterances to 8-month-olds than to 4- or 6-month-olds, presumably in response to the infant's changing level of comprehension. Mothers used more sentences with subjects, verbs, or objects deleted to 8-month-olds and more complex sentences to 4-month-olds.

Mucin gene and antigen expression in biliopancreatic carcinogenesis.

Mucins are high molecular weight glycoproteins which are heavily glycosylated with many carbohydrate side chains. In epithelial cancers such as biliopancreatic cancer, both quantitative and qualitative alterations in carbohydrate and polypeptide moieties of mucin glycoproteins occur. These changes in mucin glycoproteins are one of the most common phenotypic markers of biliopancreatic carcinogenesis and may play an important pathobiological role. The expression of some of the sialylated carbohydrate antigens appears to correlate with a poor prognosis and increased metastatic potential in biliopancreatic cancer. The increased exposure of peptide epitopes of mucin glycoproteins in biliopancreatic cancer appears to be due to either abnormal glycosylation and/or altered levels of mucin gene transcription. In addition, dysregulation of tissue specific mucin gene expression occurs in biliopancreatic cancer. This information is currently being exploited for further elucidation of the molecular mechanisms involved in carcinogenesis, tumor progression and metastasis, and the development of novel methods of diagnosis and therapy of biliopancreatic cancer.

Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family.

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.

A trisaccharide acceptor analog for N-acetylglucosaminyltransferase V which binds to the enzyme but sterically precludes the transfer reaction.

Development of inhibitors specific for the glycosyltransferases involved in the biosynthesis of asparagine-linked sugar chains has been undertaken in the hopes that these compounds may serve as tools to elucidate the roles of complex carbohydrates in biological recognition events. We report here the first example of a glycosyltransferase acceptor analog in which strategic replacement of a nonreacting hydroxyl group with a larger substituent produces a molecule which is recognized by the enzyme but does not react because of a steric block to the glycosyl transfer reaction. N-Acetylglucosaminyltransferase V catalyzes the transfer of GlcNAc from the sugar nucleotide donor UDP-GlcNAc to the 6-OH group of mannose in the synthetic trisaccharide acceptor beta GlcNAc(1-->2)alpha Man(1-->6)beta Glc-O(CH2)7CH3 (Km = 23 +/- 2 microM; Vmax = 116 +/- 3 pmol/h) to form the tetrasaccharide beta GlcNAc(1-->2)(beta GlcNAc(1-->6))alpha Man(1-->6)beta Glc-O(CH2)7CH3. The acceptor analog produced by replacement of the adjacent nonreacting 4-OH group of the mannose residue with an O-methyl group was not a substrate for the enzyme but was found to be a good competitive inhibitor of GlcNAc transferase V with Ki = 14 +/- 2 microM. To test the theory that it was the presence of the large methyl group which prevented the glycosyl transfer reaction the 4'-deoxygenated analog was synthesized. It was found to be a good substrate with Km = 74 +/- 6 microM and an almost 5-fold higher kcat (Vmax = 535 +/- 13 pmol/h). NMR data show no evidence of important conformational differences between the trisaccharide analogs, and kinetic experiments detected no differences for the binding of UDP-GlcNAc in their presence. The conclusion was therefore reached that the large methyl group introduced on O-4' sterically prevented the formation of product even though both potential substrates were bound by the enzyme. This "steric exclusion" strategy offers potential for the design of inhibitors for that class of glycosyltransferases in which the reactive hydroxyl group is also an essential recognition element.

Key involvement of all three GlcNAc hydroxyl groups in the recognition of beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp-OR by N-acetylglucosaminyltransferase-V.

N-Acetylglucosaminyltransferase-V (GlcNAc T-V) transfers a beta-linked GlcNAc residue from UDP-GlcNAc to OH-6' (of the alpha Man residue) in oligosaccharides terminating in the sequence beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp(or Manp)-OR (3, R = (CH2)7CH3). It was previously found that OH-4" (of the GlcNAc residue) in 3 was a critical element for substrate recognition by this enzyme. We show here that OH-3" and OH-6" are also key recognition elements. Four analogs of trisaccharide 3 where OH-3" and OH-6" were replaced, independently, by NH2 and NHAc groups, were prepared by multi-step chemical synthesis and kinetically evaluated as substrates for GlcNAc T-V from hamster kidney. These substitutions were selected since they replaced the OH groups with groups probing both hydrogen bonding and steric bulk. The 3"-modified compounds were found to be very poor substrates with Km values more than 50-fold elevated over that for 3 (26 microM) while the 6"-modified compounds were completely inactive. An intact 3,4,6 triol system in the terminal GlcNAc residue therefore appears to be the key polar group system that is recognized by this enzyme.

Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Galbeta1-3GalNAcalpha1-Se r/Thr); they lack core 2 O-linked glycans (Galbeta1-3(Galbeta1-4GlcNAcbeta1-6)GalNAcalpha1 -Ser/Thr) because they do not express the core 2 beta1 6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack alpha1 3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an alpha1 3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of beta-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P-selectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin. PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin.