EULAR recommendations for women's health and the management of family planning, assisted reproduction, pregnancy and menopause in patients with systemic lupus erythematosus and/or antiphospholipid syndrome.

OBJECTIVES: Develop recommendations for women's health issues and family planning in systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS). METHODS: Systematic review of evidence followed by modified Delphi method to compile questions, elicit expert opinions and reach consensus. RESULTS: Family planning should be discussed as early as possible after diagnosis. Most women can have successful pregnancies and measures can be taken to reduce the risks of adverse maternal or fetal outcomes. Risk stratification includes disease activity, autoantibody profile, previous vascular and pregnancy morbidity, hypertension and the use of drugs (emphasis on benefits from hydroxychloroquine and antiplatelets/anticoagulants). Hormonal contraception and menopause replacement therapy can be used in patients with stable/inactive disease and low risk of thrombosis. Fertility preservation with gonadotropin-releasing hormone analogues should be considered prior to the use of alkylating agents. Assisted reproduction techniques can be safely used in patients with stable/inactive disease; patients with positive antiphospholipid antibodies/APS should receive anticoagulation and/or low-dose aspirin. Assessment of disease activity, renal function and serological markers is important for diagnosing disease flares and monitoring for obstetrical adverse outcomes. Fetal monitoring includes Doppler ultrasonography and fetal biometry, particularly in the third trimester, to screen for placental insufficiency and small for gestational age fetuses. Screening for gynaecological malignancies is similar to the general population, with increased vigilance for cervical premalignant lesions if exposed to immunosuppressive drugs. Human papillomavirus immunisation can be used in women with stable/inactive disease. CONCLUSIONS: Recommendations for women's health issues in SLE and/or APS were developed using an evidence-based approach followed by expert consensus.

Prevalence of acute alcohol intoxication in Borgo Trento Hospital Emergency Department (Verona).

INTRODUCTION: Alcohol is an important factor weighting towards global disease burden, premature death and Disability-Adjusted Life Years. This study examines the burden imposed on the Borgo Trento Verona Hospital Emergency Department (ED) Italy by patients with Acute Alcohol Intoxication (AAI). METHODS: A 6-year retrospective study was performed by reviewing medical records in all patients (≥ 16 years old) diagnosed with AAI. Clinical criteria for inclusion in the sample followed those defined in the "Alcohol Intoxication Symptoms" section of DSM-IV-TR. Ambiguous cases presenting traumas potentially related to AAI were confirmed positive using a Blood Alcohol Level (BAL) test before inclusion in the sample. Socio-demographic data, case history, timing of admission/discharge and outcome were collected for each patient. RESULTS: One thousand five hundred forty-seven patients (males:females = 6.6:1) were included in the study. With regard to marital status, the crude rate within the sample shows that single and married subjects have a significantly higher prevalence (p < 0.01). When demographic data of residents was taken into consideration, divorced/separated and single categories were significantly more represented (p < 0.05). The admissions appeared fairly constant throughout the week with a higher prevalence of patients aged between 25-55 years, and a significant peak of the youngest age class on weekends. Higher rates of admission were recorded during the late afternoon and night. The triage code of admission appeared uniformly distributed along the week, with the highest prevalence of green code (67.7%), followed by yellow one (25.8%). The analysis of clinical symptoms and BAL highlights that moderate clinical severity concerns almost 50%, and clinically critical severity the 6.6% of cases of access. Admissions rates of foreign patients resulted to be 3 times higher compared to those of natives when adjusted to demographic data. DISCUSSION: Social disadvantages such as foreign provenance and social difficulties as unemployment, low money intake, perception of loneliness, and dysfunction in family life were frequently identified in the sample. An exception was found within the youngest age group, where the AAI are mostly related to alcohol abuse during social outings over the weekend. EDs play a crucial role in helping patients with AAI, but prevention of alcohol abuse though implementation of social and sanitary health policies on all ages (but especially among the youngest) is essential.

The Traveller's Risk Perception (TRiP) questionnaire: pre-travel assessment and post-travel changes.

BACKGROUND: Travellers' risk perception is a key component of travel risk assessment because it influences the adequate implementation of safety precautions. The aims of this study are to validate a tool to analyse travellers' risk perception to identify which factors can influence it and how it changes upon return. METHODS: The Traveller's Risk Perception (TRiP) questionnaire was developed and administered to outpatients before and after travel in three travel clinics. A principal component analysis (PCA) was performed to validate the questionnaire and multivariate regression analysis was used to evaluate the effect of travellers' characteristics on the risk scores. RESULTS: A total of 1020 travellers completed the questionnaire. PCA identified two latent factors: 'generic-disseminated risks' and 'specific-circumstantial risks'. Cronbach's α was acceptable (0.76 and 0.70, respectively). The 'generic-disseminated risks' dimension scored higher than the 'specific-circumstantial risks' (p<0.001). The items with the highest scores were insect bites, gastrointestinal disorders and malaria. The mean scores were significantly lower after the travel for all items but one. CONCLUSIONS: The TRiP questionnaire is a valid and reliable tool for rating travellers' perceptions. Staff in travel clinics should be trained to systematically assess travellers' risk perception in order to tailor the consultation according to specific information needs.

Near-ultraviolet difference absorption and circular dichroism studies on partially synthetic ribonucleases S'.

Near-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-,1epsilon,7epsilon-diguanidono-[Asn14]-, [Phe(F)8, Orn10]- and 1epsilon, 7epsilon-diguanidino-S-peptide, with S-protein. Environmental alterations of Phe-8 in the S-peptide and Tyr-25 in the S-protein, derived from the association process, lead to strong optical signals whose location and magnitude were clearly defined by means of a comparative analysis of the above spectra. Additionally, the spectroscopic effects resulting from insertion of a tyrosyl residue into an hydrophobic environment in the presence or absence of hydrogen-bonding partners were identified and compared with similar findings obtained from the model compound p-cresol.

Far-ultraviolet difference absorption and circular dichroism studies on partially synthetic ribonucleases S'.

Far-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-, 1epsilon, 7epsilon-diguanidino-[Asn14]-, [Phe(F)8, Orn10]-, [Cha8, Orn10]- and 1epsilon, 7epsilon-diguanidono-S-peptide, with S-protein. The aromatic chromophores contributions to the absorption spectra in the 220-250 nm wavelengths interval strongly exceed the hyperchromism due to the random coil to right handled alpha-helix transition of the S-peptide, accompanying the association process. Contributions resulting from peptide transitions constitute a large portion of the total dichroism, nevertheless substitution of the non aromatic cycloexylalanine residue in position 8 of the S-peptide with phenylalanine, p-fluorophenylalanine and tyrosine leads to CD negative maxima located at 215, 215 and 212,5 nm, respectively. The strongest ellipticity increment (28%), relative to that of the Cha-derivative, was observed at 212,5 nm for the [Tyr8]-S-peptide analog, while in the narrow 222 nm range minimal differences were found upon insertion into the position 8 of the S-peptide of the above aromatic residues. Information derived from above data and from a comparison of RNAase A, S and S-protein CD spectra enabled us to assign the positive CD band at 240 nm in RNAase A spectrum to transitions of phenylalanines and inaccessible tyrosines.

Protamines. II. Circular dichroism study of the three main components of clupeine.

The three main components YI, YII, and Z of clupeine, a protamine from herring, have been purified and characterized. The conformational preferences of clupeines have been examined as a funciton of pH, temperature, added salts, and presence of structure-disrupting agents and helix-supporting solvents using circular dichroism. It was found that these small basic proteins assume predominantly an unordered conformation in aqueous solution. Addition of counter ions, in particular perchlorate, and 2-chloroethanol induces in various amounts the onset of the right-handed alpha-helical conformation. Urea favors the statistical coil state. It was also demonstrated that in the 0.1--4.0 . 10(-1) M range, in contrast to clupeines YI and Z, the circular dichroic properties of the YII component do not seem to be sensitive to the addition of mono- and diphosphate.

Specificity determinants of maize casein kinase-IIB are related to but distinct from those of rat liver casein kinase-2.

The site specificity of maize seedling casein kinase-IIB, a type-2 casein kinase exhibiting an unusually low Mr, has been studied with the aid of model acidic peptide substrates for rat liver casein kinase-2. Like the animal enzyme, casein kinase-IIB also readily phosphorylates peptides SEEEEE, SEAEEE and SEEEAE, but not SEEAEE. Maize seedling casein kinase-IIB, however, is almost inactive toward peptides SAEEEE, SAEEEEE and SAAEEEEE which are good substrates for liver casein kinase-2. This indicates that casein kinase-IIB requires acidic residues not only at position +3, similar to rat liver casein kinase-2, but also at position +1, where the animal enzyme tolerates a neutral residue. This and other differences outlined in this report support the view that protein kinases of the same type from different sources may have significant differences in their substrate specificity.

Stimulation by NaCl, polylysine and heparin of two forms of spleen tyrosine protein kinase immunologically related with the protein expressed by lyn oncogene.

The previously isolated spleen tyrosine protein kinase, conventionally termed TPK-IIA, displaying activation by either positively or negatively charged polyelectrolytes has been further characterized. TPK-IIA is immunologically related with the tyrosine protein kinase encoded by the lyn gene, a member of src subfamily and is dramatically activated by very high NaCl concentration. The stimulatory effects of NaCl and polylysine, which are not additive, are accounted for by increased Vmax values, the Km being virtually unchanged, suggesting that both effectors probably interact with the same site(s). Stimulation of TPK-IIA by heparin appears to be partially additive to that promoted by NaCl and possibly occurring through a different mechanism. The NaCl activatory effect correlates with the electrolytic nature of synthetic peptides used as substrates, being much more consistent with neutral peptides as compared with acidic ones. Of the other three spleen tyrosine protein kinases, TPK-I shows similar biochemical and immunological features, suggestive of close relatedness with TPK-IIA, while TPK-IIB and TPK-III are neither related with the lyn protein nor with the products of three other oncogenes of the src subfamily, namely lck, hck and fyn.

Synthetic peptide substrates for casein kinase 2. Assessment of minimum structural requirements for phosphorylation.

Unlike the peptides SAEAAA and SEEAAA which are not substrates for casein kinase 2 (CK-2) their analogs SAAEAE and SAAEAA are still significantly phosphorylated. Their Km values, however, (13.3 and 18.9 mM, respectively) are almost two orders of magnitude higher than that of SEEEEE and their Vmax values are 3- and 14-fold lower than that of SAAEEE. The peptide ESEEEEE, but not ASEEEEE, is a slightly better substrate than SEEEEE, while both RSEEEEE and SEEEKE are very poor substrates compared to ASEEEEE and SEEEAE, respectively. SAAEAE is much more responsive to polylysine stimulation and polyphosphate inhibition than is SEEEEE. Taken together these data show that a single acidic residue at the third position from the C-terminal side of the phosphorylatable amino acid represents not only a necessary, but also a sufficient condition for site recognition by CK-2. Optimal phosphorylation efficiency, however, requires an extended C-terminal cluster of several acidic residues, and can be compromised by the presence of only a basic residue either inside the acidic cluster or adjacent to the N-terminal side of the phosphoacceptor amino acid. The structure of the phosphoacceptor site can greatly influence the efficacy of substrate-directed effectors of CK-2.

Further definition of the substrate specificity of the alpha-herpesvirus protein kinase and comparison with protein kinases A and C.

The pseudorabies virus protein kinase prefers model substrates containing arginyl residues on the amino-terminal side of a target seryl or threonyl residue. We have defined this substrate specificity more precisely in experiments using a new series of synthetic model peptides. When the number of arginyl residues was varied from two to four in substrates of the type RnASVA it was found that peptides with four arginyl residues constituted the best substrates, although the most marked decrease in Km was seen on increasing the number of arginyl residues from two to three. The effect of varying the number of 'spacer' alanyl residues from zero to three was investigated in peptides of the type R4AmSVA, and the peptide with one alanyl residue was found to be the best substrate, making R4X the optimal amino-terminal environment for this enzyme. A similar substrate specificity was observed with the herpes simplex type 1 protein kinase. Protein kinase C was found to have a quite similar substrate preference to the viral enzyme as far as the number and position of the amino-terminal basic residues was concerned; but, unlike the viral protein kinase, it also requires carboxy-terminal basic residues in optimal peptide substrates, and can tolerate the substitution of lysyl for arginyl residues. The cyclic AMP-dependent protein kinase, like the viral enzyme, had favourable kinetic constants for this series of peptides, but differed from the latter in being able to catalyze the phosphorylation of the peptides with two to four arginyl residues with similar efficiency. Studies with the protein, clupeine Y1, as substrate indicated that the pseudorabies virus protein kinase can tolerate arginyl residues on the carboxyl-terminal side of its target residue when there are suitable amino-terminal arginyl determinants. In this respect the virus protein kinase resembled protein kinase C but differed from the cyclic AMP-dependent protein kinase which cannot tolerate such carboxyl-terminal basic residues. The relationship of substrate specificity with model peptides to the ability of the pseudorabies virus protein kinase to phosphorylate proteins in vitro and in vivo is discussed.

An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A.

The synthetic phosphopeptide RRATpVA was found to be the most effective substrate for protein phosphatase 2C (PP2C) so far identified. Replacement of phosphothreonine by phosphoserine decreased activity over 20-fold and a striking preference for phosphothreonine was also observed with two other substrates (RRSTpTpVA and casein) that were phosphorylated on both serine and threonine. Replacement of the C-terminal valine in RRATpVA by proline abolished dephosphorylation, while exchanging the N-terminal alanine by proline had no effect. The preference for phosphothreonine and the effect of proline are similar to protein phosphatase 2A (PP2A). However, the peptide RRREEETpEEEAA, an excellent substrate for PP2A, was not dephosphorylated by PP2C, and substitution of the C-terminal valine in RRATpVA by glutamic acid reduced the rate of dephosphorylation by PP2C over 10-fold, without affecting dephosphorylation by PP2A. Addition of two extra N-terminal arginine residues to RRASpVA increased PP2A catalysed dephosphorylation 4- to 5-fold, without altering dephosphorylation by PP2C. These results represent the first study of the specificity of PP2C using synthetic peptides, and strengthen the view that this approach may lead to the development of more effective and specific substrates for the serine/threonine-specific protein phosphatases.

The substrate specificity of the protein kinase induced in cells infected with herpesviruses: studies with synthetic substrates [corrected] indicate structural requirements distinct from other protein kinases.

Synthetic peptides have been used to investigate the site specificity of highly purified virus induced protein kinase, a recently discovered protein kinase isolated from cells infected with alpha-herpesviruses. The enzyme from cells infected with pseudorabies virus can catalyse the phosphorylation of both seryl and threonyl residues in peptides that contain several arginyl residues on the amino-terminal side of the target residue. At least two arginyl residues are required, and the best substrates examined contain four to six such residues. Virus induced protein kinase differs in site specificity from protein kinase C in being unable to phosphorylate peptides in which multiple arginyl residues are on the carboxyl-terminal side of the target residue, or to phosphorylate peptides in which the arginyl residues are replaced by ornithyl residues. Virus induced protein kinase from cells infected with herpes simplex virus type I had similar substrate preferences to virus induced protein kinase from cells infected with pseudorabies virus. Although virus induced protein kinase and the cyclic AMP-dependent protein kinase have several peptide substrates in common, their relative preferences for these (as indicated by Km values) were found to be very different.

Isolation and partial characterization of distinct forms of tyrosine protein kinases from rat spleen.

Three peaks of tyrosine protein kinase activity (TK-I, TK-II and TK-III) can be resolved when the extract of rat spleen particulate fraction is subjected to DEAE-cellulose gradient chromatography. TK-I and TK-II, insensitive to both EGF and insulin, have been further purified by Sephacryl S200 gel filtration and characterized. TK-I has an apparent mR of 65000, by far prefers Mn2+ over Mg2+ as activator, can use GTP besides ATP as phosphate donor and is stimulated 2-3-fold by polylysine. TK-II, whose mR approximates 50000, is equally activated by Mg2+ and Mn2+, does not use GTP and is insensitive to polylysine. TK-I and TK-II can phosphorylate the synthetic peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly (as well as its derivative with Orn in place of Arg), angiotensin II and poly(Glu, Tyr) 4:1 which exhibits different km values with TK-I and TK-II, (100 and 10 microM, respectively). When TK-I was incubated with [gamma-32P]ATP and MnCl2 a doublet of alkali-stable radiolabeled bands with molecular masses of 55 and 60 kDa were observed. Under identical conditions TK-II gives rise to a single alkali-stable radiolabeled band of 51 kDa, which may represent the autophosphorylation product of TK-II itself.

Opposite and mutually incompatible structural requirements of type-2 casein kinase and cAMP-dependent protein kinase as visualized with synthetic peptide substrates.

The synthetic hexapeptide Ser-Glu-Glu-Glu-Val-Glu and its N-acetylated derivative are readily and specifically phosphorylated by rat liver casein kinase TS (type-2), while the derived heptapeptide with an additional N-terminal Arg is a very poor substrate. Conversely, the substitution of Glu for Val5 in the synthetic peptide Arg-Arg-Ser-Thr-Val-Ala, which is a good substrate for cAMP-dependent protein kinase by virtue of the N-terminal arginyl residues, prevents its phosphorylation by this enzyme. These data indicate that the site specificities of these two classes of protein kinases, requiring acidic and basic residues on the C- and N-terminal sides of the target residue(s), respectively, are mutually incompatible.

Polyglutamyl peptides: a new class of inhibitors of type-2 casein kinases.

Casein kinase-TS (Ck-TS), a type-2 casein kinase purified from rat liver cytosol which phosphorylates seryl and threonyl residues N-terminal to acidic clusters, is specifically inhibited by polyglutamyl peptides which are ineffective both on type-1 casein kinase and on cAMP-dependent protein kinase. The inhibition is competitive toward the protein substrate and non-competitive toward ATP. Among the polyglutamates tested (Glu)70 is the most effective (Ki 0.11 microM). (Glu)10 and (Glu)5 are also inhibitors, though less powerful than (Glu)70, while (Glu)3, (Glu)2 and free glutamic acid up to 5 mM are ineffective. These results disclose the possibility that naturally occurring polypeptides containing long stretches of acidic residues may act as physiological inhibitors of type-2 casein kinases.

Distinct structural requirements of Ca2+/phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase as evidenced by synthetic peptide substrates.

Protein kinase C, purified to near homogeneity from the brain, has been tested toward a variety of synthetic peptide substrates including different phosphorylatable residues. While it proved totally inactive toward the tyrosyl peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly, as well as toward several more or less acidic seryl peptides, it phosphorylates with a Ca2+/phospholipid-dependent mechanism, at seryl and/or threonyl residues, many basic peptides, some of which are also good substrates for cAMP-dependent protein kinase (A-kinase). Among the peptides tested, however, the best substrate for protein kinase C, with kinetic constants comparable to those of histones, is the nonapeptide Gly-Ser-Arg6-Tyr, which is not a substrate for A-kinase. Moreover, although the peptide Pro-Arg5-Ser-Ser-Arg-Pro-Val-Arg is a good substrate for both kinases, its derivative with ornitines replacing arginines is phosphorylated only by protein kinase C. Some typical substrates of A-kinase on the other hand, like the peptides Phe-Arg2-Leu-Ser-Ile-Ser-Thr-Glu-Ser and Arg2-Ala-Ser-Val-Ala, are phosphorylated by protein kinase C rather slowly and with unfavourable kinetic constants. It is concluded that, while both protein kinase C and A-kinase need basic groups close to the phosphorylatable residues, their primary structure determinants are quite distinct.