Ratio of triglycerides to HDL cholesterol is an indicator of LDL particle size in patients with type 2 diabetes and normal HDL cholesterol levels.

OBJECTIVE: In patients with type 2 diabetes, a normal HDL cholesterol level does not rule out that LDL particles may be small. Although techniques for analyzing LDL subfractions are not likely to be used in clinical practice, a prediction of LDL size based on a regular lipid profile may be useful for assessment of cardiovascular risk. RESEARCH DESIGN AND METHODS: Sixty patients with type 2 diabetes with acceptable glycemic control and an HDL cholesterol level > or = 1 mmol/l were recruited after cessation of lipid-altering treatments. LDL size was determined by 2-20% PAGE; patients having small LDL (n = 30) were compared with those having intermediate or large LDL (n = 30). RESULTS: Clinical characteristics, pharmacological therapies, lifestyle, and prevalence of diabetes-related complications were similar in both patient groups. LDL size correlated negatively with plasma triglycerides (TGs) (R2 = 0.52) and positively with HDL cholesterol (R2 = 0.14). However, an inverse correlation between the TG-to-HDL cholesterol molar ratio and LDL size was even stronger (R2 = 0.59). The ratio was > 1.33 in 90% of the patients with small LDL particles (95% CI 79.3-100) and 16.5% of those with larger LDL particles. A cutoff point of 1.33 for the TG-to-HDL cholesterol ratio distinguishes between patients having small LDL values better than TG cutoff of 1.70 and 1.45 mmol/l. CONCLUSIONS: The TG-to-HDL cholesterol ratio may be related to the processes involved in LDL size pathophysiology and relevant with regard to the risk of clinical vascular disease. It may be suitable for the selection of patients needing an earlier and aggressive treatment of lipid abnormalities.

Aminopeptidase B (EC 3.4.11.6).

Aminopeptidase B (EC 3.4.11.6) is a Zn(2+)-dependent exopeptidase which selectively removes arginine and/or lysine residues from the NH2-terminus of several peptide substrates including Arg0-Leu-enkephalin, Arg0-Met-enkephalin and Arg-1-Lys0-somatostatin-14. Analysis of its primary structure showed that aminopeptidase-B is structurally related to leukotriene A4 hydrolase, an important enzyme of the arachidonic acid pathway. This structural relationship is further supported by the capacity of aminopeptidase-B to hydrolyse leukotriene A4. Aminopeptidase-B is widely distributed in a number of tissues, including endocrine and non-endocrine cells. Moreover, in rat PC12 pheochromocytoma cells, the enzyme is secreted and associated with the external face of the plasma membrane. Together these data strongly argue in favour of a role of this bi-functional enzyme in the final stages of precursor processing mechanisms occurring either in the secretory pathway, at the plasma membrane, or at both locations.

Restriction maps for the cottontail rabbit herpesvirus (CTHV) genome.

The sites for restriction endonucleases ApaI, BamHI and PvuII in the genome of the cottontail rabbit herpesvirus were localized. The physical mapping of the 150-kb DNA was facilitated by peculiarities of the genome structure, namely the presence of repetitive DNA and of invertible segments, and by the analysis of overlapping cosmid clones.

N-arginine dibasic convertase (NRD convertase): a newcomer to the family of processing endopeptidases. An overview.

N-arginine dibasic convertase (NRD convertase) (accession number L27124) is a metalloendopeptidase from rat brain cortex and testis which cleaves peptide substrates on the N-terminus of arginine residues in basic doublets. Its predicted amino acid sequence contains the putative zinc binding motif HXXEH in a region which exhibits 35% and 48% similarity with E coli protease III (pitrilysin E.C 3.4.99.44) and rat or human insulinase (E.C 3.4.99.45) respectively. This feature clearly classifies this endopeptidase as a member of the pitrilysin family of zinc-metalloproteases. However, the NRD convertase sequence contains a distinctive additional feature consisting of a 71 acidic amino acid stretch. Its substrate selectivity and the characteristic motifs of its amino acid sequence allow us to propose this new metalloendopeptidase as the first member of a new class of processing enzymes.

Sequence of the canine herpesvirus thymidine kinase gene: taxon-preferred amino acid residues in the alphaherpesviral thymidine kinases.

Multiple sequence alignments of evolutionarily related proteins are finding increasing use as indicators of critical amino acid residues necessary for structural stability or involved in functional domains responsible for catalytic activities. In the past, a number of alignments have provided such information for the herpesviral thymidine kinases, for which three-dimensional structures are not yet available. We have sequenced the thymidine kinase gene of a canine herpesvirus, and with a multiple alignment have identified amino acids preferentially conserved in either of two taxons, the genera Varicellovirus and Simplexvirus, of the subfamily Alphaherpesvirinae. Since some regions of the thymidine kinases show otherwise elevated levels of substitutional tolerance, these conserved amino acids are candidates for critical residues which have become fixed through selection during the evolutionary divergence of these enzymes. Several pairs with distinctive patterns of distribution among the various viruses occur in or near highly conserved sequence motifs previously proposed to form the catalytic site, and we speculate that they may represent interacting, co-ordinately variable residues.

Possible relation between the U54 segment of the CTHV genome and the conserved gene block C rearranged in alpha and gamma herpesvirus genomes.

The genome of CTHV is an atypical member of the gamma-2 subgroup of herpesvirus genomes that contains two segments (instead of one) of DNA with low G+C content flanked by highly repetitious DNA with high G+C content. The segments freely undergo polarity inversion with respect to one another. We have found nucleotide sequences in one of these segments, the U54 segment, whose putative translational products show clear similarity to two ubiquitous herpesvirus gene products, a single-stranded DNA binding protein and a protein of the helicase superfamily. These sequences are located within 5 kilobase pairs of the ends of the segment, suggesting that U54 may be related to a genetically defined entity (gene block C; Davison and Taylor (1987) J. Gen Virol. 68, 36-48) issuing from a previous sequence comparison of the gamma-1 genome of Epstein-Barr virus and the alpha genome of varicella-zoster virus.

Aminopeptidase-B in the rat testes: isolation, functional properties and cellular localization in the seminiferous tubules.

An aminopeptidase of the B-type, with an apparent M(r) 72,000 and pI = 4.9, was isolated from rat testes and characterized. The enzyme was able to remove only Arg and/or Lys residues from L-amino acid beta-naphthylamide derivatives and from the N-terminus of several peptides. No cleavage occurred in the case of Arg-Pro bonds as found in bradykinin and substance P. The enzyme was sensitive to cysteinyl reagents and to aminopeptidase inhibitors, such as bestatin, amastatin and arphamenines A and B. The aminopeptidase activity, tested with L-Arg beta-naphthylamide and with Arg0-Met-enkephalin as substrates, was inhibited by o-phenanthroline, and restored by Zn2+ suggesting its metallopeptidase character. The partial characterization of an aminopeptidase-B activity in rat brain cortex identified a protein which is biochemically and immunologically related to the testis enzyme. By immunohistochemistry, the aminopeptidase-B was found to be particularly abundant in the seminiferous tubules at late stages of spermatogenesis and was clearly detected in a restricted area of elongated spermatids. Remarkably, the enzyme was observed to concentrate massively in the residual bodies. Since this aminopeptidase-B was able in vitro to trim out N-terminal Arg and/or Lys residues from peptides mimicking processing intermediates, it is proposed that this enzyme may be involved in propeptide and proprotein processing mechanisms in the course of spermatid differentiation.

Plasma fatty acid status in Moroccan children: increased lipid peroxidation and impaired polyunsaturated fatty acid metabolism in protein-calorie malnutrition.

In previous studies on plasma fatty acid and antioxidant status in 29 malnourished Moroccan children (12 with mild protein-calorie malnutrition, 17 with severe protein-calorie malnutrition) compared to 15 healthy control children from the same area, we pointed out that these populations were heterogeneous in terms of their essential fatty acid and antioxidant status. The aim of the present study was to classify the children using the Waterlow classification and their essential fatty acid status. The discrepancies in lipid parameters, nutritional and inflammatory markers, blood oxidative indexes, antioxidant micronutrients or trace elements (selenium, zinc, vitamin E) related to polyunsaturated fatty acids were checked in these populations. Eight of the control subjects and nine of the severe protein-calorie malnutrition children were essential fatty acid-deficient, compared to only one of the mild protein-calorie malnutrition group. Examination of the essential fatty acid-sufficient subjects with mild protein-calorie malnutrition, compared to the essential fatty acid-sufficient control subjects, showed only a decrease in Z scores and a non-significant decrease in selenium and vitamin E. In severely malnourished children, albumin, cholesterol and low density lipoprotein (LDL) cholesterol, plasma selenium, vitamin E and zinc were low, whereas inflammatory proteins and triglycerides were high. These features worsened with essential fatty acid deficiency. In all protein-calorie malnutrition subjects, there was oxidative stress (increase in thiobarbituric-acid reactants, imbalance between plasma polyunsaturated fatty acid, vitamin E and selenium levels), even in the absence of essential fatty acid deficiency. Monounsaturated fatty acids, oleic acid/stearic acid (C18:1 n-9/C18:0) delta9 desaturase and n-3 and n-6 elongase activity indexes increased. The C18:1/C18:0 delta9 desaturase activity index was negatively correlated to Z scores (r = -0.44, P< 0.01 for Z score weight, r = -0.39, P < 0.01 for Z score height), albumin (r = -0.82, P < 0.01) and zinc (r = -0.51, P< 0.01) levels. In essential fatty acid-deficient, severe protein-calorie malnutrition subjects, delta6 desaturase activity was impaired, and there was a non-significant decrease in arachidonic acid. Essential fatty acid deficiency is a type of malnutrition, and is associated with an aggravation of all parameters in severe protein-calorie malnutrition. The increase in the C18:1/C18:0 delta9 desaturase activity and enhanced lipid peroxidation without any essential fatty acid deficiency could be early markers of protein-calorie malnutrition.

Lipids, lipoproteins, and fatty acids during infantile marasmus in the Fès area of Morocco.

The lipid composition of plasma, including total HDL and LDL cholesterol, triglycerides, apo AI, apo B, and fatty acids was investigated in 29 malnourished Moroccan children in two groups: 12 children with mild PCM, and 17 with severe PCM. Normally nourished children from the same area (n = 15) served as controls. The severe malnourished children showed a significant reduction of apo AI, total and LDL cholesterol, and an increase in the levels of triglycerides. Furthermore, these children showed a decrease in the saturated fatty acids myristic and stearic acid, and a similar decrease in the essential fatty acid (EFA) metabolites, especially eicosatrienoic acid, arachidonic acid, and eicosapentaenoic acid, with an increase in the oleic and cisvaccenic monounsaturated fatty acids. In contrast, the PCM group showed only an increase of docosatetraenoic and docosapentaenoic, with an associated decrease in myristic acid and palmitic acid. On the other hand, the indexes of delta 9 desaturase and elongase n-3 and n-6 were increased, and this was found to be related to the severity of the malnutrition. These results suggest that the severity of malnutrition is associated with an increase of desaturation and elongation of PUFA, EFA deficiency and/or peroxidation.

Rapid detection of pseudorabies virus genomic sequences in biological samples from infected pigs using polymerase chain reaction DNA amplification.

The presence of the pseudorabies virus (PRV) genome in infected hosts has previously been studied by standard hybridization techniques, which showed the viral genome to be present at very low levels in infected tissues. The recently introduced polymerase chain reaction (PCR) procedure provides an alternative and rapid means of amplifying small quantities of specific DNA sequences. We applied this technique to a study of pigs infected by PRV. The sequence selected for amplification consisted of 222 base pairs lying in the gene coding for the glycoprotein gp50. We used a pair of 20-mer oligonucleotides flanking this sequence as primer and a cloned Stu-Nde fragment containing the sequence as target DNA. To avoid the tedious DNA extraction procedure we performed PCR directly on disrupted cells and detected specific amplification after 25 cycles of PCR with the thermostable Taq DNA polymerase. Amplified products were detected by gel electrophoresis directly. Nasal samples from experimentally and naturally infected pigs were tested by this PCR technique. When compared with tissue culture and serological tests, detection by gel electrophoresis of PCR amplified fragments provided excellent specificity and sensitivity. We concluded that PCR amplification will be a valuable tool for rapid diagnosis of PRV infection in pigs, taking less than 1 h to complete.

[Herpesviridae: classification and structure in 1991]. / Herpesviridae: classification et structure en 1991.

In 1981, herpesviruses were classified by the International Committee of Taxonomy of Viruses (ICTV, 1) inside the herpesviridae family. Progress in biotechnology and molecular biology during the last 10 yr, has permitted the characterization of new viruses and genomic structures. The objective of this paper is to collect the data found in the literature since 1981, to actualize the description of herpesviridae family.

Effects of two low-dose oral contraceptives containing ethinylestradiol and either desogestrel or levonorgestrel on serum lipids and lipoproteins with particular regard to LDL size.

This study was designed to determine the effects of two low-dose oral contraceptives, most frequently given in our area, monophasic desogestrel/ethinylestradiol (DG/EE) and triphasic levonorgestrel/ethinylestradiol (LNG/EE), on lipoprotein parameters, especially LDL particle size and HDL subclass distribution (determined by lipid-stained 2%-20% polyacrylamide gradient gel electrophoresis) in 37 healthy normolipidemic women aged 19 to 27 years. Lipid and lipoprotein parameters were measured before the start of treatment and in the third month of oral contraceptive use. Results reflected the estrogen-progestin balance. As compared with baseline values, with both formulations, plasma total cholesterol, phospholipids, and HDL3 cholesterol increased, and LDL-predominant peak size decreased, with a translation of LDL pattern A towards pattern I. With DG/EE, plasma triglycerides, apolipoproteins AI and B increased. With LNG/EE, LDL cholesterol increased, and HDL2 cholesterol decreased. All these modifications were moderate, within threshold limits. Estrogen-dominant monophasic DG/EE appears to be more favorable than progestin-dominant triphasic LNG/EE, since the reduction in LDL-predominant peak size is not associated with an increase in LDL cholesterol or with a decrease in HDL2 cholesterol.

Effect of an acute zinc depletion on rat lipoprotein distribution and peroxidation.

The aim of this study was to determine the extent to which zinc depletion leads to lipoprotein modifications by measuring both lipoprotein-fraction distribution and peroxidation in zinc-depleted rats. The animals were divided into three groups and fed for 8 wk a zinc-adequate diet (100 ppm) ad libitum (AL), a zinc-deficient diet (0.2 ppm) ad libitum (ZD), or a zinc-adequate diet according to the pair feeding method (PF). Trace-element status, tissular lipids, and lipoprotein-fraction study were performed. The MDA production by the lipoprotein fraction was measured before and after induced peroxidation. Cholesterol and phospholipids were increased in ZD rats. An important increase of VLDL and IDL was observed and a significant enhanced production of MDA by the LDL was related to zinc deficiency. From this observation, we may conclude that LDL fractions of ZD rats are more susceptible to induced oxidative damage. These results suggest that in zinc deficiency, the lipoprotein fragility is an aggravating factor of peroxidation and the dyslipoproteinemia may lead to an atherogenic risk.

[Plasma apolipoproteins AI and B: reference values in the Isère population and determination of individual values]. / Apolipoprotéines AI et B plasmatiques: valeurs usuelles de la population de l'lsère et détermination de valeurs seuil.

Reference ranges for apolipoprotein AI and B plasma concentrations were established in a population of unrelated apparently healthy volunteers (138 men and 186 women) living in the region of Grenoble. Apolipoproteins were measured using an immunoturbidimetric assay on a Cobas Fara II analyzer, with reagents and IFCC standardized calibrators from Orion. Apolipoprotein AI mean concentration was higher in women than in men and increased with age in both men and women older than 45. Apolipoprotein B mean concentration was higher in men and increased linearly with age in both sexes. Linear regression analysis was used to determine desirable and high risk values for apolipoproteins AI and B from the guidelines developed by the National Cholesterol Education Program for HDL cholesterol and LDL cholesterol, respectively. Our data indicate that an apolipoprotein AI value of 1.05 g/l is comparable to an HDL cholesterol value of 0.35 g/l. The apolipoprotein B cutpoints of 1.15 g/l for men and 1.05 g/l for women correspond to the accepted LDL cholesterol cutpoint of 1.60 g/l.

[Effects of oral contraceptive preparations and smoking on lipoprotein repartition]. / Effets de la contraception orale et du tabac sur la répartition des lipoprotéines.

We studied the effect of oral contraceptives and smoking on the lipid profile of 251 women and 72 men, 20-29-year-old. In women, taking estroprogestatives, cholesterol, triglycerides, apoproteins AI and B were higher than in controls; HDL-cholesterol was not modified. Lipoprotein analyses in polyacrylamide gradient gel exhibited an increase of the HDL3 fraction at the expense of the HDL2 fraction, with a reduced LDL size. Smoking in addition to estroprogestative absorption accentuated these modifications and led to a decreased HDL-cholesterol (HDL2 fraction essentially), with an increased LDL-cholesterol. In men, smoking resulted in higher levels of total cholesterol, apoprotein B and LDL-cholesterol, without any significant change in LDL size, higher levels of triglycerides and lower level of the HDL2 fraction without any change in HDL-cholesterol. In women, smoking led only to an increase in triglycerides. In summary, analysis of the distribution of HDL subclasses and of LDL size showed an evolution towards a supposed more atherogenic lipid profile in women taking oral contraceptives associated or not with smoking, and in male smokers.

A bovine cell line stably expressing porcine tumor necrosis factor alpha (TNF-alpha): growth properties and permissivity for pseudorabies virus replication.

To explore the feasibility of modifying the pathogenicity of pseudorabies virus (PrV), we have undertaken a program to develop recombinant PrV carrying the porcine tumor necrosis factor alpha (TNF-alpha) gene. We have used a cloned genomic DNA fragment containing the entire porcine TNF-alpha gene (Chardon et al., 1991), and have deleted the 3' non-translated region which is supposed to be a tissue-specific regulatory sequence. We have also removed the 5' non-translated region containing a TATA-box and binding sites for other transcription factors. The resulting TNF-alpha cassette has been inserted into a neomycin-selectable shuttle vector. This construct has been used to select MDBK cell lines harbouring the TNF-alpha-carrying plasmids in order to verify the production of biologically active TNF-alpha. One cell line thus obtained secreted 20-30 pg/10(6) cells of active TNF-alpha after five days in culture and exhibited normal growth kinetics. PrV titers on this cell line were the same as titers on cells not expressing TNF-alpha, indicating that TNF-alpha expression in MDBK cells does not abrogate their permissivity for virus replication. Our results show that construction of recombinant PrV expressing TNF-alpha should be possible using the MDBK cell line as host.

NRD convertase and aminopeptidase B: two processing metallopeptidases with a selectivity for basic residues.

An endoprotease and an aminopeptidase B were isolated from rat testis and characterized. The first one is a metalloendopeptidase of 1161 residues which contains a canonical HXXEHX76E Zn(2+)-binding site and an acidic stretch of 71 amino acids containing 79% of Glu and Asp. It exhibits an in vitro selectivity for peptide bonds at the N-terminus of Arg (R) moieties in dibasic sites and was thus called NRD convertase (Nardilysin: EC 3.4.24.61). It belongs to the pitrilysin family and shows 24 and 34% identity with E. coli protease III (EC 3.4.24.54) and insulysin (EC 3.4.24.55) respectively. The aminopeptidase B component is a 72 kDa metalloexopeptidase which is able to remove Lys and Arg residues from naphtylamide derivatives and from the N-terminus of various peptide substrates. A combination of biochemical and immunochemical studies revealed its ubiquitous character. In the testis, both enzymes are highly expressed at late stages of spermatogenesis and NRD convertase expression is exclusively restricted to the germ cells. The subcellular localization of both enzymes supports the involvement of aminopeptidase B in processing events associated with the secretory pathway but led to new hypothesis on the possible physiological role(s) of NRD convertase.