Histological investigation for comparison of cartilaginous tumors of unknown biological course with unequivocal chondrosarcomas.

Morphological findings recorded from chondromatous tumors of unknown biological behaviour (n = 40) were compared with those obtained from unequivocal chondrosarcomas (n = 27), and the histological results were evaluated by clinical outcome. The morphological details were analysed by histological set up and summarized by Mirra et al. (1985): Two benign patterns, the enchondroma encasement pattern and the island of cartilage patterns, were the most common findings in cartilaginous tumors of unknown biological behaviour. Malignant patterns, such as chondrosarcoma permeation, invasion of the Haversian system, soft tissue mass and bands of fibrosis patterns were detected only in unequivocal chondrosarcomas. However, the enchondroma encasement pattern could be focally demonstrated also in central chondrosarcomas (n = 3), a phenomenon, which in our opinion could not be interpreted as being indicative of a preexisting benign lesion. Morphological classification of chondromatous tumors of unknown biological behaviour, as a group of benign cartilaginous tumors, enables their discrimination from unequivocal chondrosarcomas. This was also confirmed by the significant difference (p = 0.0065; Mantel-Cox) among survival curves after Kaplan and Meier (1958). We conclude that the above criteria, when applied with due caution, are helpful in classifying chondromatous tumors.


Information analysis of immune and endocrine organs. Morphological changes in the course of infection.

Morphological and morphometric studies were conducted into lymphoid and endocrine organs of 259 human adults and infants with pyoinflammatory diseases (PID) and of 300 experimental mice. Informative and correlation analyses of the data thus recorded provided evidence to the effect that in the course of an infection process adaptation and compensation responses were characterized by intensified exchange of information within the immune-endocrine system (IES). Septic courses of PID were found to be accompanied by impairment of inter-organ correlations, increase in information entropy and progressive structural disorganization of the IES.


HTLV-I associated gastric lymphoma.

A large gastric lymphoma was detected in a 42-year-old woman who was seropositive for HTLV-I. The tumor was histologically shown to be of diffuse large cell type, and phenotypic analysis revealed high expression of CD3 and CD4, indicative of a helper/inducer type of T-cell lymphoma. In Southern blot analyses, the tumor cells showed T-cell receptor re-arrangement and contained proviral DNA of HTLV-1.


[Classification of causes of death in humans].

The cause of death of 2631 deceased persons (2418 adults, 213 children) was determined. Classification is proposed by monocausative, relatively monocausative, competitive and complex causes of death. This classification would be in accordance with types of dying, as suggested by Thieke and Nizze [17]. A comparison of results has revealed far-reaching agreement between the two principles of classification with regard to frequency distribution of causes, except for the category of complex causes which, however, was of minor importance in our investigations. This was attributed to the fact that classification was facilitated by consideration of epistemological approaches. Differences among categories of causes were attributable also to age and sex of the cases studied, both adults and children.


[Capillarosclerosis of the lower urinary tract and chronic alcohol consumption].

Between 1984 and 1987, autopsies were performed on 214 decreased who were safely assumed to have been chronic consumers of alcohol. Histological investigations were conducted on 84 ureters, with twelve of them (14.3 percent) exhibiting capillarosclerosis. This statistical relationship between alcohol consumption and capillarosclerosis was not attributable to action of alcohol on the capillaries but most probably had been a consequence of polytoxicomania, e.g. chronic alcohol abuse in concomitance with intake of phenacetin-containing medicaments. Accidental post-mortem finding of capillarosclerosis may be interpreted as a relative indicator to chronic alcohol consumption.


Polygenic influences on the length of oestrous cycles in inbred mice involve MHC alleles.

Genetic influences on female reproductive cycles were analysed in histocompatibility-congenic strains of mice. Oestrous cycles of young, virgin mice of inbred-congenic strains, hybrid crosses (F1), and parental-hybrid backcrosses (F2) were monitored for 3 months. Oestrous cycles were categorized by length (inter-oestrous interval): 4, 5, 6, or 7-14 days. Mice with the following H-2 haplotypes had a greater proportion of 5-day oestrous cycles: H-2b, H-2r, H-2h2, H-2h4, and H-2i5. In contrast, the H-2k and H-2d haplotypes had mostly 4-day oestrous cycles. Influences of H-2 haplotype were seen on two genetic backgrounds, C57BL/10Sn and C3H. Non-H-2 alleles were also implied by different patterns of cycles between strains with the same H-2b haplotype: C57BL/10Sn with predominantly 5-day cycles vs. C57BL/6J with a mix of 4- and 5-day cycles. The genetic basis for strain differences was investigation in F1 hybrids and their backcrosses. F1 hybrids of an H-2b (C57BL/10Sn; 5-day cycles) and an H-2k (B10.BR; 4-day cycles) strain had mostly 5-day cycles, indicating dominance of an H-2b allele(s). However, F1 hybrids from the reciprocal B6 x B10 cross (both H-2b) also display a preponderance of 5-day cycles, indicating dominance of a non-H-2 autosomal allele from the C57BL/10Sn strain. Among F2 mice, a '4-day' phenotype segregated with homozygosity for the k haplotype (P < 0.05, chi 2). These findings demonstrate the influence of genetic differences at the major histocompatibility complex on oestrous cycles.


DNA typing of HLA-DQ alleles by gene amplification of DQA and DQB variable exons: analysis of DQA/DQB haplotypes.

In the present report, we describe a DNA typing method that allows detection of all the polymorphic variants of DQA1 and DQB1 second exons. By the oligotyping procedure provided in this paper, we are able to identify 8 DQA1 and 13 DQB1 alleles and to type random individuals in any heterozygous combination. We provide the hybridization and washing temperatures for using either 32P labelled or non-radioactive probes. The discrimination power of this procedure, compared to serological and cellular techniques, is remarkable. Therefore, this typing method finds perfect application in transplantation immunology and it will be very helpful to optimize the matching of unrelated donors before BMT. It is apparent from our results that despite the linkage disequilibrium present between DQ and DR loci, a DR specificity may frequently be associated to different DQ haplotypes. This is the case for DR4, DR7, DR8, DR9, and DR13 specificities.


The HLA-DRB1*0405 haplotype is most strongly associated with IDDM in Algerians.

Insulin-dependent diabetes mellitus (IDDM) in Caucasians is strongly associated with HLA-DR3-DQ2 and DR4-DQ8. In order to investigate the HLA class II associations with IDDM in Algerians, we have used polymerase chain reaction (PCR) and sequence specific oligonucleotide analysis (SSO) to identify DQA1, DQB1, and DRB1 alleles, haplotypes and genotypes in 50 unrelated IDDM patients and 46 controls from a homogeneous population in Western Algeria. Both DRB1*0301-DQA1*0501-DQB1*0201 (DR3-DQ2) and DRB1*04-DQA1*0301-DQB1*0302 (DR4-DQ8) haplotypes were found at increased frequencies among the patients compared to controls (45% vs. 13%, RR = 5.5, Pc < 10(-5) and 37% vs. 4%, RR = 12.9, Pc < 10(-4), respectively). Among the latter, in contrast to other Caucasian populations, only DRB1*0405-DQA1*0301-DQB1*0302 was significantly increased in the Algerian patients (25% vs. 1% in controls, RR = 30.3, Pc < 10(-3). Accordingly, the highest risk of disease was observed in DRB1*0301-DQA1*0501-DQB1*0201/DRB1*0405-DQA1+ ++*0301-DQB1*0302 heterozygotes (34% in patients vs. 0% in controls; RR = 49; Pc < 10(-3). This observation and its comparison with DR-DQ haplotypes in other ethnic groups suggest that the DRB1*0405 allele which encodes an Asp57-negative beta chain may contribute to IDDM susceptibility in a similar way as Asp57-negative DQ beta chains.


One-step molecular HLA-DR prescreening employing a set of 14 sequence specific oligonucleotides in a non-radioactive tetramethylammonium chloride hybridization protocol.

A novel non-radioactive protocol for molecular generic HLA-DR typing is introduced, employing sequence specific oligonucleotides (SSOs) enzymatically 3'-labelled with biotin-14-dATP via terminal deoxynucleotidyltransferase in a tetramethylammonium chloride hybridization procedure. The detection reaction is carried out, using streptavidin conjugated horseradish peroxidase which is bound to the SSOs, in combination with a light emitting detection system. Fourteen SSOs and one control SSO are employed for generic HLA-DR typing in a one-step protocol. In order to demonstrate the suitability of this procedure, 5 homozygous typing cell lines and samples of 11 pretyped individuals which include most serologically defined HLA-DR specificities (DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9, 10, 52, and DR53) are analysed with the panel of 14 SSOs. The typing results show that this protocol, which avoids the use of radioisotopes, combines high specificity and easy handling. It also allows typing of poorly amplified samples because even after longer exposition times no false positive signals were observed and is particularly suitable for routine molecular HLA-DR typing on the generic level. In addition it can easily be adapted to DP and DQ typing or DR subtyping.


Diallelic polymorphism may explain variations of the blood concentration of mannan-binding protein in Eskimos, but not in black Africans.

Mannan-binding protein (MBP) is a lectin which, upon binding to certain carbohydrates, activates the classical pathway of complement without the involvement of antibody or C1q. Deficiency of the MBP is associated with an opsonic defect and recurrent infections during early life. An amino acid substitution in the exon 1 at codon 54 in the MBP gene (GGC [glycine] to GAC [aspartic acid]) has been shown to be closely associated with low MBP concentration in Caucasoids. The gene frequency of the mutant allele in this population has been estimated at 0.13. In the study described here, we investigated the association between the mutant allele and MBP protein concentration in Eskimos from East-Greenland and black Africans from the Baringo District in Kenya. The frequency of the GAC allele was identical in Eskimos and Caucasoids (0.13). No overlap with regard to MBP concentration between the genotypes was found in the Eskimos. In contrast, the Africans revealed a low frequency of the GAC allele (0.009). However, the median MBP protein concentration was approximately 5 times lower among the Africans than the Eskimos. In 12.6% of the Africans and in 2.5% of the Eskimos, MBP was undetectable. Thus, MBP deficiency is the most frequent immunodeficiency so far described. The high prevalence of MBP deficiency among healthy individuals indicates that MBP deficiency also confers some selective advantages. We advance the hypothesis that MBP deficiency is maintained in populations because MBP deficiency decreases the infectivity of some intracellular micro-organisms which are dependent on opsonization.


C5 deficiency in A/J mice is not associated with resistance to the development of secondary amyloidosis.

The aim of the study was to determine whether C5 deficiency in the mouse is associated with resistance to the development of secondary amyloidosis. Chronic inflammation was induced in the F2 progeny, derived from matings between amyloid-susceptible and amyloid-resistance mice, by daily injections of azocasein for thirty days. Using a restriction fragment length polymorphism generated by digestion of genomic DNA with the restriction enzyme HindIII, C5 sufficient and deficient DNA can be clearly differentiated. Eight mice were found to be C5 sufficient, 32 were heterozygotes and 14 were found to be C5 deficient. Grading of the splenic amyloid load from negative to 4+ was performed after staining tissue squashes with Congo red and viewing them under a polarizing microscope. Seventeen mice were noted to have negative to trace, 18 had moderate (1+ - 2+) and 19 had heavy (3+ - 4+) amyloid deposition. There was no correlation between splenic amyloid load and C5 deficiency. Based on these results it is clear that C5 deficiency and resistance to secondary amyloidosis are not associated.


The removal of the type-2 copper from Rhus vernicifera laccase.

We have studied the removal of the type-2 copper from tree laccase (Rhus vernicifera) by treatment with EDTA at pH 5.2 in the presence of a redox buffer containing ferri- and ferrocyanide. The efficiency with which the copper is removed depends on the Fe(CN) 6(4-)/Fe(CN) 6(3-) ratio. We have varied this ratio from approx. 2:1 to about 50:1 and the best results were obtained with the highest ratio, i.e., the most cathodic solution potential. Nevertheless, the presence of Fe(CN) 6(3-) is required for the procedure to be effective. Although we cannot exclude the possibility that a mixed-valence form of laccase is the reactive species, we believe the results are better explained by a model which assumes that the removal of the type-2 copper depends upon an ordered sequence of oxidation-reduction reactions. Specifically, we propose that the copper is released as the monovalent ion from previously reduced laccase and then reoxidized in solution and sequestered with EDTA. The reoxidation step drives the reaction because recombination with the protein is inhibited when copper is in the divalent form. In testing this model, we have also shown that the type-2 copper can be removed under strictly reducing conditions when 4,4'-dicarboxy-2,2'-biquinoline (BCA) is present to complex the copper(I) ion. Although the BCA method is effective, the reaction takes longer, perhaps because of the limited solubility of BCA at the pH values of interest. Finally, we have found that the best results are obtained with either method when a cyanometalate ion such as Fe(CN) 6(3-) or Co(CN) 6(3-) is present in the medium. The exact role of this factor has yet to be established, but there is no indication that free cyanide has a role in the process. The most likely interpretation is that some type of binding interaction with the protein facilitates copper release.


Kinetic properties of type-II ATP diphosphohydrolase from the tunica media of the bovine aorta.

The kinetic properties of type-II ATP diphosphohydrolase are described in this work. The enzyme preparation from the inner layer of the bovine aorta, mostly composed of smooth muscle cells, shows an optimum at pH 7.5. It catalyzes the hydrolysis of tri- and diphosphonucleosides and it requires either Ca2+ or Mg2+ for activity. It is insensitive to ouabain (3 mM), an inhibitor of Na+/K(+)-ATPase, to tetramisole (5 mM), an inhibitor of alkaline phosphatase, and to Ap5A (100 microM), an inhibitor of adenylate kinase. In contrast, sodium azide (10 mM), a known inhibitor for ATPDases and mitochondrial ATPase, is an effective inhibitor. Mercuric chloride (10 microM) and 5'-p-fluorosulfonylbenzoyl adenosine are also powerful inhibitors, both with ATP and ADP as substrates. The inhibition patterns are similar for ATP and DP, thereby, supporting the concept of a common catalytic site for these substrates. Apparent Km and Vmax, obtained with ATP as the substrate, were evaluated at 23 +/- 3 microM and 1.09 mumol Pi/min per mg protein, respectively. The kinetic properties of this enzyme and its localization as an ectoenzyme on bovine aorta smooth muscle cells suggest that it may play a major role in regulating the relative concentrations of extracellular nucleotides in blood vessels.


Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.


A novel alpha-type fibrinogenase from Agkistrodon rhodostoma snake venom.

By means of CM-Sephadex C-50 column chromatography, gel-filtration on sephadex G-75 and Sephacryl S-200 columns, a purified fibrinogenase, kistomin, was obtained from venom of Agkistrodon rhodostoma. It was a single peptide-chain with a molecular mass of about 21,800 Da containing about 202 amino-acid residues as revealed by amino acid analysis. Kistomin preferentially cleaved A alpha- and subsequently the gamma-chain of fibrinogen, leaving the B beta-chain unaffected. Its fibrinogenolytic activity was estimated to be 36.6 +/- 4.5 mg/min per mg protein and was inhibited by the pretreatment of EDTA, suggesting that it is a metalloproteinase. Its fibrinogenolytic activity in platelet-poor plasma is much less potent as compared to that in purified fibrinogen solution. It inhibited ristocetin-induced aggregation of human platelets in a dose-dependent manner in the presence of von Willebrand factor.


Quantitation of molybdopterin oxidation product in wild-type and molybdenum cofactor deficient mutants of Chlamydomonas reinhardtii.

A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120 degrees C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway.


Three matrix metalloproteinases form a non-covalent association with the rhoptry-associated protein-1 of Plasmodium falciparum.

During the characterization of malaria vaccine candidate proteins, three metalloproteinases having a molecular mass of 220, 95 and 70 kDa were found to be co-isolated with the rhoptry-associated protein-1 (RAP-1) complex, but not with RAP-3 or gp195. These enzymes were also found in detergent extracts of saponin-lysed Plasmodium falciparum. Of nine proteinase inhibitors tested, only EDTA was found to abrogate activity. Dose-dependent curves were determined for several metal ions and cobalt was found to synergistically enhance enzyme activity. The gelatinases were immunoprecipitated with monospecific polyclonal antisera to macrophage and fibroblast gelatinase; however, these sera did not react with intracellular parasites by indirect immunofluorescence. These results indicate that the matrix metalloproteinases co-isolated with RAP-1 originate from human serum used to cultivate P. falciparum in vitro.


Molecular modeling of the 3-D structure of cytochrome P-450scc.

Sequence-alignment studies of the bovine mitochondrial cholesterol side-chain cleavage enzyme cytochrome P-450scc with the bacterial cytochrome P-450cam (camphor hydroxylating enzyme) have been undertaken. Our novel alignment of the sequences revealed 69 identical residues and many highly conserved regions. The results of the sequence alignment studies were used to model the 3-D structure of P-450scc based on the available crystal structure of P-450cam. The major insertions in the sequence are found mainly on four external-loop regions of the molecule, while the core structure of P-450cam is retained with subtle internal modifications. The most hydrophobic of these four external loops is proposed as a candidate for membrane attachment.


Purification and amino-acid sequence of a nerve growth factor from the venom of Vipera russelli russelli.

Nerve growth factor (NGF) was purified from the venom of Vipera russelli russelli by Sephadex G-50 gel filtration, S-Sepharose column chromatography and Blue-Sepharose CL-6B column chromatography. The purified NGF was found to be a glycoprotein, whose apparent molecular mass was estimated to be about 17.5 kDa by SDS-PAGE. The amino-acid sequence was determined by a combination of conventional methods. The V. r. russelli NGF was composed of 117 amino-acid residues with one residue, Asn-21, being N-linked glycosylated and the molecular mass of its protein portion was calculated to be 13,280 Da.


Characterization of cardiac Na+/Ca(2+)-exchange by site-directed polyclonal antibodies.

Cardiac Na+/Ca(2+)-exchange is an integral membrane protein consisting of approx. 970 amino acids with as many as 12 membrane-spanning and 11 extramembranal regions (Nicoll, D.A., Lognoni, S. and Philipson, K.D. (1985) Science 250, 562-565). Based upon primary sequence information, 3 amino-acid sequences located in either extramembranal segment a or f, consisting largely of acidic amino-acids, were selected for the production of synthetic peptides. The peptides were cross-linked to carrier ovalbumin and used to generate site-directed polyclonal antibodies (sd-Ab). Western blot analysis of bovine cardiac sarcolemmal (SL) proteins demonstrated that sd-Ab against segment a and 1 against loop f recognized a 70 kDa protein and a lower molecular mass band at 55 kDa under reducing conditions. A different loop f sd-Ab failed to recognize the 70 kDa protein but did associate with a 120, 65 and 55 kDa protein under the same conditions. Under non-reducing conditions, antibodies to all three peptides recognized the 65 kDa protein. All sd-Ab were blocked by addition of their respective peptides and were not inhibited by either of the other peptides. A sd-Ab against loop f was immobilized to an affinity support matrix and used to immunoprecipitate detergent solubilized cardiac SL vesicle protein. Immunoprecipitated protein was reconstituted into proteoliposomes which demonstrated Na+/Ca(2+)-exchange activity. Immunoprecipitated protein cross-reacted with sd-Ab against all three peptides with bands at 120, 70 and 55 kDa on Western blots. Tryptic digests of native SL vesicles abolished recognition of segment a sd-Ab for SL proteins while having little or no affect on reactivity to the protein by both sd-Ab against loop f. Digestion of the SL vesicle protein with endoproteinase Arg C did not alter sd-Ab recognition. The results suggest that specific domains of the cardiac Na+/Ca(2+)-exchanger depending upon the conformation of the protein, may not be available for antibody binding. The 70 kDa polypeptide appears to include the N-terminal region of the protein and what is believed to be a large cytoplasmic extramembranal loop. Limited proteolysis by trypsin and endoproteinase Arg C yielded results consistent with the model which places the N-terminus of the protein on the extracellular surface and a large extramembranal segment (loop f) on the cytoplasmic side of the SL membrane.


Phosphofructokinase from white muscle of the rainbow trout, Oncorhynchus mykiss: purification and properties.

Phosphofructokinase was purified and characterized from the white skeletal muscle of rainbow trout Oncorhynchus mykiss. Purification involved three steps: ion-exchange chromatography on hydroxyapatite and affinity chromatography on phosphocellulose and ATP-agarose. A final specific activity of 75 units per mg of protein at 22 degrees C and pH 7.2 with 40% recovery was obtained. The purified enzyme gave a single band on SDS-PAGE with a subunit molecular mass of 76.5 +/- 0.6 kDa. Based on gel filtration analysis, the active form of the enzyme was found to be composed of six identical subunits. A high isoelectric point (7.1) was found for this enzyme. Arrhenius plots of the enzyme activity showed a sharp transition at 15-16 degrees C. The pH optimum of the enzyme was 8.0-8.5 at physiological level of ATP and positive modulators shifted the optimum to lower pH values. Amino-acid analysis revealed a lower content of the aromatic residues Phe, Tyr and Trp and higher level of Ser residue than in the rabbit muscle enzyme.


Human serum amyloid P-component (SAP) selectively binds to immobilized or bound forms of C-reactive protein (CRP).

The two homologous human pentraxins, C-reactive protein (CRP) and serum amyloid P-component (SAP), specifically bind to each other only when the CRP is in an immobilized form bound to one of its ligands or to an antibody. CRP did not bind to immobilized SAP. The binding of SAP to immobilized forms of CRP was Ca(2+)-dependent and of sufficient affinity to occur in the presence of serum or purified serum proteins. SAP bound preferentially to a synthetic peptide corresponding to the Ca(2+)-binding region of CRP. Monoclonal antibodies to a synthetic peptide corresponding to the Ca(2+)-binding region selectively inhibited the binding interaction. Proteolytic cleavage of CRP between residues 146 and 147 within the Ca2+ binding region abolished the SAP-binding site; however, the intact subunits of the pentameric CRP were capable of binding SAP. The significance of the binding interaction is that it may serve as the basis for localization of SAP to sites of tissue damage or repair, sites where CRP is selectively deposited.


Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).


Reversible interactions between plasminogen activators and plasminogen activator inhibitor-1.

We have shown that the urokinase (UK) kringle domain contains a high-affinity plasminogen activator inhibitor-1 (PAI-1) binding site, responsible for the 10-fold faster complex formation between UK and PAI-1 than between PAI-1 and low-molecular-weight urokinase (LMWUK). Complex formation between UK and PAI-1, but not between LMWUK and PAI-1, was suppressed 10-fold in the presence of peptide U-107 derived from the UK kringle domain. Peptide U-373 derived from the UK catalytic domain slowed complex formation between UK and PAI-1 and also LMWUK and PAI-1. Inactivation of tissue-type plasminogen activator (tPA) by PAI-1 was slowed 10-fold in the presence of peptides derived from the tPA finger and kringle-2 domains. DFP-inactivated (DIP) UK and both forms of DIP-tPA inhibited PAI-1 binding to U-107 and to U-373 whereas single-chain urokinase-type PA (scuPA) was unable to compete with either peptide for PAI-1 binding. These data suggest that the reversible PAI-1 binding site in the UK A-chain plays a role in the rapid association with PAI-1 as important as those that reside in the tPA A-chain and that reversible PAI-1 binding sites are expressed on the surface of UK upon conversion from scuPA, in contrast to tPA.


Mass spectrometry in the integrated strategy for the structural analysis of protein variants.

A significant proportion of genetic disorders are caused by point mutations in proteins, and many variants of haemoglobin have been characterized by mass spectrometry. The strategy employs fast atom bombardment and/or tandem mass spectrometry of enzymatically digested peptides for elucidating the type and position of the mutations. Electrospray ionization mass spectrometry is useful for detecting mutations in the intact protein molecule, but the competence diminishes with increasing mass or increasing number of 13C atoms. Recent developments of DNA techniques requires us to re-evaluate the status of mass spectrometry in this field. The role of mass spectrometry in the structural analysis of protein variants is discussed, and is compared with chemical methods and DNA strategies.


Validation of a gas chromatographic/mass spectrometric method for the determination of (E)-beta-fluoromethylene-m-tyrosine and its active metabolite in human plasma and urine.

(E)-beta-Fluoromethylene-m-tyrosine (FMMT, MDL 72394) represents a prodrug approach to site-selective, irreversible inhibition of monoamine oxidase. A sensitive and specific method for the quantification of FMMT and its active metabolite (MDL 72392) in human plasma and urine has been developed for future pharmacokinetic studies. The procedure consists of a liquid-liquid extraction of plasma and urine samples, esterification with HCl/butanol and, subsequently, N-acylation with pentafluoropropionic anhydride. This is followed by gas chromatography/mass spectrometry employing negative ion chemical ionization. The precision and the accuracy of the method have been optimized by using two internal standards: MDL 72661 (a methyl homologue of FMMT) for the quantification of FMMT, and MDL 72761 (a methyl homologue of MDL 72392) for the quantification of MDL 72392. The method has been validated for FMMT and MDL 72392 over the concentration range 2.5-400 pmol ml-1 with a limit of quantification for both compounds of 2.5 pmol ml-1. Sample clean-up and selected ion monitoring by mass spectrometry ensured the specificity and sensitivity required for the pharmacokinetic evaluation of FMMT and MDL 72392.


Fast atom bombardment, electron ionization and chemical ionization mass spectrometry of 1,2,3-benzenetricarboxylic acids, inhibitors of the mitochondrial tricarboxylate carrier.

It has been discovered that 1,2,3-benzenetricarboxylic acid and some 5-substituted derivatives are specific and competitive inhibitors of the mitochondrial tricarboxylate carrier. In order to characterize these acids a study was carried out using electron ionization (EI), chemical ionization (CI) and fast atom bombardment (FAB) mass spectrometry on the free acids, the potassium salt of 1,2,3-benzenetricarboxylic acid and the corresponding methyl esters. Unimolecular decomposition processes were also studied using B/E linked scan experiments in order to correlate peaks present in the FAB mass spectra of the above compounds. The FAB ionization method, as expected, was the most suitable for analysing the very polar and thermolabile acids and the only method which could be used for the analysis of the potassium salt. EI and CI turned out to be appropriate methods for analysing the less polar and thermostable trimethyl esters.


Characterization of the molecular species of glycerophospholipids from rabbit kidney: an alternative approach to the determination of the fatty acyl chain position by negative ion fast atom bombardment combined with mass-analysed ion kinetic energy analysis.

An alternative approach to identifying fatty acid chain position in the molecular species of glycerophospholipids has been studied and developed. The fatty acyl groups esterified to the glycerol backbone in isomeric glycerophosphatidyl-choline, -serine and -ethanolamine as well as glycerophosphatidic acid can be detected by the presence of a pair of anions derived from phosphatidic acid parent ions (M minus the polar head groups in glycerophospholipids), designed to be [M--polar head--R2COOH]- and [M--polar head--R2CO--H]-, produced by negative ion fast atom bombardment combined with mass-analysed ion kinetic energy analysis. Because of the significant abundance of [M--polar head--R2COOH]- anion, fatty acid chains differing by 2 Da can be distinguished by accurate measurements of the electrostatic voltage related to this ion. Three-volt differences can be evidenced. Using this approach, the molecular species of glycerophosphatidyl-choline, -serine, -ethanolamine and -inositol from rabbit kidney were characterized after the separation of both class and species by normal and reversed-phase high-performance liquid chromatography, respectively. We identified 11 arachidonoyl-containing molecular species of glycerophospholipids and the other 17 lipid molecules in this biological material. A couple of 1- alkenyl-2-arachidonoyl-sn-glycerol-3-phosphoethanolamine species, identified as plasmalogen GPE 16:0-20:4 and plasmalogen GPE 18:0-20:4, were found for the first time in rabbit kidney.


Determination of erythromycin A in salmon tissue by liquid chromatography with ion-spray mass spectrometry.

A reverse-phase liquid chromatography/mass spectrometry (LC/MS) method, incorporating gradient elution, is described for the characterization of residual erythromycin A and its metabolites in salmon tissue. The method uses ion-spray, a mild atmospheric pressure ionization technique which provides an abundant protonated molecule well suited for selected ion monitoring experiments. Tandem mass spectrometry (MS/MS) using collision-induced dissociation was used to provide structural information. The LC/MS method was tested for the analysis of salmon tissue spiked with erythromycin A at levels between 0.01 and 1 p.p.m. A simple extraction and clean-up procedure, slightly modified from that described by Takatsuki et al. (J. Assoc. Off. Anal. Chem. 70, 708 (1987)), was used in this work. Using selected ion and selected reaction monitoring techniques, the LC/MS and LC/MS/MS methods provided detection limits of < 10 and 50 ng g-1, respectively. Confirmatory full-scan LC/MS and LC/MS/MS spectra were obtained at the 0.5 and 1 microgram g-1 levels, respectively. Using a combination of these techniques, the presence of residual erythromycin A was confirmed in the tissue of fish administered medicated feed containing the antibiotic. In addition, several metabolites and degradation products of erythromycin A, including anhydro-erythromycin and N-demethyl-erythromycin, were detected and where possible confirmed by comparison with authentic compounds. Although this analytical method has been shown to afford the necessary sensitivity and precision, application of these techniques to high-throughput quantitative analyses will require development of an improved clean-up procedure and preferably also of a suitable surrogate internal standard.


On-line high-performance liquid chromatographic/gas chromatographic/tandem ion trap mass spectrometric determination of levamisole in milk.

On-line high-performance liquid chromatography/high-resolution gas chromatography/tandem mass spectrometry, performed on a quadrupole ion trap, has been used as a rapid method for the quantification of the anthelmintic drug levamisole in raw milk extracts following a simple extraction procedure. Detection at the 0.5 p.p.b. level and a linear response in the 10-0.5 p.p.b. range is demonstrated. Multiple-scan monitoring techniques have been used to acquire chemical ionization tandem mass spectra and electron impact mass spectra in a single chromatographic run.


Two-dimensional electrophoresis of urine specimens from patients with renal disease.

In an effort to develop non-invasive markers capable of characterizing renal disease with greater sensitivity and specificity than those currently available, urinary proteins were studied by high-resolution two-dimensional electrophoresis in order to catalog those proteins which appeared to be most affected by a variety of renal diseases. Urine specimens were prepared by high-pressure liquid chromatography and subjected to two-dimensional gel electrophoresis. Proteins were visualized with silver stain. Gels from 17 patients were analyzed in detail and compared to standard maps of human urinary proteins and plasma. The two-dimensional gels displayed approximately 100 times the number of proteins demonstrated by high resolution agarose electrophoresis. We have identified 34 proteins whose urinary concentrations were most affected by renal disease. Twenty seven were of plasma origin, based upon comigration with plasma samples (17 of unknown identity and function). Seven proteins were observed in normal urine but not present in plasma. These proteins, which may represent kidney tissue proteins, were not apparent in the urine of patients with proteinuria, most likely due to dilutional effects.


Monoclonal gammopathy in a 30 weeks old premature infant.

The occurrence of monoclonal gammopathy in childhood is extremely rare. This report describes the presence of a monoclonal immunoglobulin in a 30 week old premature infant, incidentally discovered by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) during an ongoing study of the plasma/serum protein development. Comparative analysis of the electrophoretogram of the infant with 'reference' protein maps revealed the presence of an 'abnormal' immunoglobulin light chain spot. A spot having an identical apparent molecular weight and isoelectric point was also detected after 2D-PAGE of the mother's plasma and its Protein-A purified immunoglobulin fraction. The observation of a monoclonal gammopathy in a premature infant, most likely transmitted from his mother, highlights the potential usefulness of 2D-PAGE in the clinical laboratory.


Rapid and automated characterisation of seed genotype using Micrograd electrophoresis and pattern-matching software.

New precast microgels are described for use in quickly identifying seed of cereal varieties by determining protein composition within an hour. For example, gliadin proteins are extracted from crushed wheat grain, wheatmeal or flour with ethylene glycol (centrifugation not necessary) and 5 microliters extract is applied to a Micrograd gel (3-15% gel gradient) for ten minutes' electrophoresis at 300 volts in sodium lactate buffer (pH 3.1). Alternatively, precast gels are available for SDS gel electrophoresis for examining a different aspect of grain composition as a means of identification. To further expedite identification, software packages have been developed to match the protein pattern for an unknown sample against those of authentic samples, thus to provide quick and definite identity, based on electrophoretic banding, densitometer scan, HPLC profile, multiple antibody reaction or RFLP pattern (PatMatch program). Furthermore, the program WhatWheat offers advice on the best combination of methods to use for a specific task of identification.


Three electrophoretic techniques comparison for des-gamma-carboxyprothrombin detection.

Des-gamma-carboxyprothrombin (DCP) is a marker that appears in the blood when modifications of vitamin K-dependent proteins carboxylation cycle occur. About 280 human plasma samples of diverse origins were tested by three different electrophoretic techniques for the evaluation of DCP: rocket immunoelectrophoresis (RIE) before and after barium carbonate adsorption, crossed affinoimmunoelectrophoresis (CAIE) and polyacrylamide gel electrophoresis in presence of calcium lactate followed by immunoblotting (PAGE-blot). A good correlation was found between CAIE and PAGE-blot in the CAIE detection limit, but not between RIE and the two other techniques. PAGE-blot was more sensitive than RIE and CAIE and allowed reliable quantification of abnormal prothrombin in plasma.


Western blotting of transforming growth factor beta 2. Optimization of the electrophoretic transfer.

The highest reported detection sensitivity of reduced monomeric transforming growth factor (TGF) beta 2 by Western blotting is 50 ng. The biologically active TGF-beta 2, which is the non-reduced dimeric protein, is even more difficult to detect by this technique. The low sensitivity is due to the poor electrophoretic transfer of the protein from the polyacrylamide gel to the blotting matrices under standard transfer conditions. By studying the effects of different blotting matrices, transfer cells, blotting buffers and their methanol content, current settings and the electrotransfer time, we have established optimal conditions for the blotting of this protein. The pH of the buffer and the blotting time are the most important factors which influence the electrotransfer yield. Optimal transfer of the TGF-beta 2 protein was achieved on PVDF membrane with semi-dry transfer for 4 h at 9 V, using 10 mM CAPS, pH 11.0, containing 5% methanol as transfer buffer. Combined with the use of a commercial antibody and an immunoblot assay kit, our optimised blotting method can detect 2 ng of the dimeric TGF-beta 2.


Red blood cell protein map: a comparison between carrier-ampholyte pH gradient and immobilized pH gradient, and identification of four red blood cell enzymes.

The aim of this study was (a) to establish a red blood cell (RBC) protein map with immobilized pH gradient for the first dimension (b) to compare the pattern with previously published RBC protein map obtained with carrier-ampholyte pH gradients and (c) to localize four new enzymes on the map (i.e. 6-phosphogluconic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glutathione peroxidase and superoxide dismutase). This publication provides the most updated RBC polypeptide pattern with twelve proteins or enzymes localized on the map.


Two-dimensional polyacrylamide gel electrophoretic characterization of proteins from organs of C3H mice expressing the scurfy (sf) genetic mutation during early and late stages of disease progression.

Scurfy (sf), is an X-linked recessive lethal mutation that occurs spontaneously in the C3H mouse. The disease is characterized by lymphoid and hematopoietic dysfunction. Affected males are of small stature and exhibit scaliness and crusting of the eyelids, ears, tail, and feet, marked splenomegaly, moderate hepatomegaly, enlarged lymph nodes, and atrophy of the thymus. The average lifespan of the affected hemizygous males (sf/y) is 24 +/- 0.7 days. Total cellular proteins were extracted from pooled samples of thymus and spleen obtained from combined litters of mice. Tissue-specific protein profiles characteristic of either sf mutant or normal mice were analyzed by two dimensional polyacrylamide gel electrophoresis (2DPAGE) at different stages of the phenotypic expression of the sf mutation, to identify changes in protein patterns that might be associated with the progression of the disease. The resultant gels were silver stained, digitized, and analyzed, by image analysis utilizing a pipelined image processor connected to a host computer. At 14 +/- 1 days of age, protein patterns from sf mutant and normal mice control organs showed considerable homogeneity, although there were proteins identified unique to the sf mutant and to the normal controls. At 20 +/- 1 days of age, the pattern differences between the sf mutant and normal control increased markedly. Differences were expressed as the percent of proteins that were unique to either the sf mutant or the normal control from the total number of each type. The percent of proteins that increased or decreased in the three organs utilized in this study ranged between 21%-39% at 14 days and were between 25%-54% at 20 days. Differences in protein expression between the normal and sf mutant as the disorder progressed for each of the three tissues examined. In addition, thymus protein profiles from 9 day old littermates that were phenotypically normal but genotypically unknown were evaluated to determine if marker proteins could be identified for the sf mutation. Limited protein changes were noted at relative molecular weights of 66, 60, 54, 39, 37, 33, 25, 23, 27, and 11 kDa. These data suggest that the sf mutation follows a trackable pattern of protein expression and repression different than the normal control C3H mouse. Several potential marker proteins associated with the sf mutation were identified in 9 day thymus prior to the phenotypic expression of the disease. These putative biomarkers may be useful for characterizing the sf mutation and the mutant may act a possible model the Wiskott-Aldrich syndrome (WAS).


Yeast 5S rRNA binding to ribosomal protein L1a alters the fluorescence of tryptophan residues lying outside the binding site.

The yeast ribosomal protein L1a contains two tryptophan residues located at positions 95 and 183. Spectrofluorometric analysis showed that the average tryptophan environment is moderately polar. Quenching studies of the yeast 5S rRNA-L1a protein complex (RNP) with acrylamide and iodide revealed tryptophan heterogeneity. The two tryptophan residues are located in the non-RNA-binding region of the L1a molecule. However, dissociation of the yeast 5S rRNA-L1a protein RNP complex to its components resulted in a decline of tryptophan fluorescence. The observation implied that the environment of the tryptophan-containing L1a regions which were not known to be involved in RNA binding was influenced by association with the 5S rRNA molecule.


Secondary structure at the beginning of the poly(A) sequence of mouse beta-actin messenger RNA.

A portion of the beta-actin mRNA of mammalian cells is believed to lack a poly(A) tail, because of its failure to bind to oligo(dT)-cellulose. S1 mapping and Northern blot analysis of this mRNA shows it to contain a poly(A) sequence of about 60 nucleotides. Only about 20-40 nucleotides are available for interaction with oligo(dT). The rest is masked, presumably by base-pairing with a poly(U) stretch present in the 3' non-coding region of the mRNA. A similar configuration occurs in the bulk of the actin mRNA, which carries a poly(A) tail with sizes ranging from approximately 60 to 200 nucleotides.


Isolation and sequence of the murine Fgf6 cDNA.

We have studied the structure of the murine Fgf6 gene encoding a fibroblast growth factor with the purpose of looking for putative regulatory sequences in the 5' and 3' non-coding regions. The Fgf6 cDNA contains a very long 3' untranslated portion of 4015 nucleotides.


Deviant energetic metabolism of glycolytic cancer cells.

The central glycolytic and oxidative pathways and the ATP-producing mechanisms differ in sane and malignant cells by their regulation and dynamics. Fast-growing, poorly-differentiated cancer cells characteristically show high aerobic glycolysis. In the same way, cholesterol biosynthesis, which occurs by normal pathways in tumors, is deficient in feed-back regulation and in sterol-transport mechanisms. Other metabolic ways are deficient, as for example, intramitochondrial aldehyde catabolism, at the origin of a possible acetaldehyde toxicity, which can be circumvented by the synthesis of an unusual and neutral product for mammalian cells acetoin, through tumoral pyruvate dehydrogenase. If most of the glycolytic pyruvate is deviated to lactate production, little of the remaining carbons enter a truncated Krebs cycle where citrate is preferentially extruded to the cytosol where it feeds sterol synthesis. Glutamine is the major oxidizable substrate by tumor cells. Inside the mitochondrion, it is deaminated to glutamate through a phosphate-dependent glutaminase. Glutamate is then preferentially transaminated to alpha-ketoglutarate that enters the Krebs cycle. Glutamine may be completely oxidized through the abnormal Krebs cycle only if a way of forming acetyl CoA is present: cytosolic malate entering mitochondria is preferentially oxidized to pyruvate + CO2 through an intramitochondrial NAD(P)(+)-malic enzyme, whereas intramitochondrial malate is preferentially oxidized to oxaloacetate through malate dehydrogenase, thus providing a high level of intramitochondrial substrate compartmentation. These and other regulatory aberrations in tumor cells appear to be reflections of a complex set of non-random phenotypic changes, initiated by expression of oncogenes.


Effects of high glucose concentration on survival and respiration of human endothelial cells.

To determine whether or not endothelial cell survival was decreased after incubation with high glucose concentrations in culture media, we studied the influence of D-glucose or L-glucose (a non-metabolizable stereoisomer of D-glucose) on cell survival using the trypan-blue exclusion test. Simultaneously, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay was used to measure both the mitochondrial respiratory chain activity and cell viability. Respiratory chain activity per cell increased when D-glucose concentrations rose but at the same time trypan-blue excluded cells were decreased. Comparison with data in the literature showed that the MTT assay was not reliable for studies involving endothelial cell survival when glucose reduction was affected on these cells. It seems important to check MTT assay reliability carefully when it is used for drugs affecting glucose metabolism, or with other cell types.


Alterations in microsomal and plasma membranes during liver regeneration.

Investigations have been carried out on the alterations of membrane lipids and some enzyme activities during liver regeneration. The results indicated that 32 h after partial hepatectomy the membrane phospholipids per mg protein were augmented. The cholesterol esters were also increased in both microsomal and plasma membranes. The specific radioactivity of the separate phospholipid fractions, estimated by incorporation of 14C-palmitate into the phospholipid molecules, was higher in membranes from partially hepatectomized rats, compared to sham-operated ones, indicating an enhanced phospholipid synthesis. The content and specific radioactivity of diacylglycerols and triacylglycerols was enhanced in both types of membranes from regenerating liver. Moreover, we observed a fluidization of these membranes, which is illustrated by the decrease of the structural order parameter (SDPH) of the lipid bilayer as well as by the elevation of the excimer to monomer fluorescent ratio (IE/IM). 1,6-Diphenyl-1,3,5-hexatriene and pyrene were used as fluorescent probes for determination of the membranes physical state. Palmitoyl-CoA and oleoyl-CoA synthetase, acyl-CoA: lysophosphocholine and acyl-CoA:lysophosphoethanolamine acyltransferase as well as phospholipase C activities were augmented in membranes from partially hepatectomized rats. The biological significance of these alterations in the process of liver regeneration is discussed.


Effect of experimental hypothyroidism on the control of 6-phosphofructo-1-kinase activity in rat jejunal mucosa.

Changes in the activity of 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) from the epithelial cells of rat small intestine during experimental hypothyroidism were studied. Hypothyroidism resulted in significant decreases in the plasma concentrations of total tri-iodothyronine, free tri-iodothyronine, total thyroxine, free thyroxine and insulin. These changes were associated with a significant increase in the plasma concentration of thyrotropin. The total activity and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (v0.5/V)) of jejunal PFK of hypothyroid rats were significantly diminished as compared to control rats. PFK of hypothyroid rats was more sensitive to inhibition by ATP. The mucosal enzyme of both control and hypothyroid state was sensitive to stimulation by AMP and fructose 2,6-bisphosphate. It is concluded that during hypothyroidism the rate of glycolytic pathway in the small intestine is reduced as a result of a fall in glucose uptake, and the subsequent kinetic changes of PFK are primarily to maintain the concentrations of fructose 6-phosphate (and glucose 6-phosphate) during the reduced glycolytic flux. These changes in PFK activity may be caused by changes in plasma insulin concentrations, glucose utilization and fructose 2,6-bisphosphate concentrations.


Self-regulation of respiratory sinus arrhythmia.

Respiratory sinus arrhythmia (RSA)--the peak-to-peak variations in heart rate caused by respiration--can be used as a noninvasive measure of parasympathetic cardiac control. In the present study four strategies to increase RSA amplitude are investigated: (1) biofeedback of RSA amplitude, (2) biofeedback of RSA amplitude plus respiratory instructions, (3) respiratory biofeedback, and (4) respiratory instructions only. All four procedures produce a significant increase of RSA amplitude from the first physiological control trial compared to baseline. This increase is faster for the groups that received respiratory biofeedback and respiratory instructions only than for the two groups that received biofeedback of RSA amplitude, the increases being equivalent for the four groups in the third session. All subjects of the group that received biofeedback of RSA amplitude only reported respiratory strategies in order to achieve the increase in RSA. Possible clinical implications of these results for parasympathetic cardiac control and cardiovascular disorders are discussed.


Self-regulation of slow cortical potentials in psychiatric patients: schizophrenia.

Slow cortical potentials (SCPs) are considered to reflect the regulation of attention resources and cortical excitability in cortical neuronal networks. Impaired attentional functioning, as found in patients with schizophrenic disorders, may covary with impaired SCP regulation. This hypothesis was tested using a self-regulation paradigm. Twelve medicated male schizophrenic inpatients and 12 healthy male controls received continuous feedback of their SCPs, during intervals of 8 s each, by means of a visual stimulus (a stylized rocket) moving horizontally across a TV screen. The position of the feedback stimulus was a linear function of the integrated SCP at each point in time during the feedback interval. Subjects were required to increase or reduce negative SCPs (referred to pretrial baseline) depending on the presentation of a discriminative stimulus. The correct response was indicated by the amount of forward movement of the feedback stimulus and by monetary rewards. Schizophrenics participated in 20 sessions (each comprising 110 trials), while controls participated in 5 sessions. Compared with the healthy controls, schizophrenics showed no significant differentiation between negativity increase and negativity suppression during the first sessions. However, in the last 3 sessions, patients achieved differentiation similar to controls, demonstrating the acquisition of SCP control after extensive training.


Morphological study on the stomach of the lesser mouse deer (Tragulus javanicus) with special reference to the internal surface.

The stomach of the lesser mouse deer (Tragulus javanicus) was observed macroscopically. It consisted of only three compartments, rumen, reticulum and abomasum without omasum. The rumen was S-shaped with large ventral and caudoventral blind sacs and the reticulum was larger than the abomasum. Internally, the rumen was covered with numerous ruminal papillae even on the pillars and the ruminoreticular fold. These papillae were leaf- or tongue-like shaped and varied in size and density. The reticulum had honey-combed crests and the secondary crests were found rarely. The lips of the reticular groove were prominent and more developed in the aboral part than in the oral one. A sac-like transition zone, which had more prominent mucosal folds than had the floor of the reticular groove, was observed between the caudal end of the reticular groove and the abomasum. Mucosal folds of the abomasum were spiral, low but rather thick. These findings were discussed in view of comparison with other ruminants and of possible functional implications.


[Complementary artificial nutrition in kidney failure].

Denutrition of the uremic patient is a substantial contributing factor to the high rate of morbimortality. At the present time there are no resources which slow the catabolic situation conditioned by humoral or hormonal factors, but resources are available which act on the nutritional factors. The aim of this paper is to study the effect of additional administration of a complete enteral diet, but high in calories and in proteins, on the nutrition of dialysis patients. Six patients undergoing periodic hemodialysis and without metabolic illness were selected: 236 ml of the solution was administered orally on a daily basis for two months. They were studied statistically using the SIGMA Program, with application of the comparison of paired averages, the variations of anthropometric and analytical data and urea kinetics. The results revealed a significant body weight increase, from 58 to 60 kg, and of the tricipital fold from 10 to 12 cm: both variations were statistically significant (p < 0.01). There were no variations in the analytical data, or in the urea kinetics (the PCR was 0.8 g/kg/day and the Kt/V was 0.8). It is concluded that it significantly improves the nutritional state, there are no side effects and it does not detract from the efficacy of the dialysis. Therefore, and although the indication for which it was designed was for predialysis patients, we think that those under periodic hemodialysis--and, form the same reasons, acute kidney failure patients--might be areas for the use of this product.


Changes of splenic lymphocyte subpopulation in mice inoculated with Babesia microti and Babesia rodhaini.

Changes of splenic lymphocyte subpopulation after Babesia microti and Babesia rodhaini inoculation in mice were examined by flow cytometric analysis. The B. microti inoculated mice showed a longer period of time from inoculation to the onset of increase or decrease parasitaemia (%), packed cell volume, total spleen cell numbers and surface immunoglobulin positive splenic cell numbers than respective periods in B. rodhaini inoculated mice. The Thy-1 positive cell numbers in B. microti inoculated mice and B. rodhaini inoculated mice pre-immunized with homologous parasites were significantly higher than that of B. rodhaini inoculated mice. The ratio of L3T4 positive cell/Lyt-2 positive cell after inoculation with B. microti was quite similar to that in B. rodhaini mice pre-immunized. However, the ratio in B. rodhaini inoculated mice revealed a lack of an increasing phase. These results suggested that the T-cell dependent early immune response, especially suppressor activity, was closely related to the difference in the course of infection between the non-lethal B. microti and the lethal B. rodhaini infection in mice.


[Control over the bacteriological quality of preparations for TPN by the sequential sampling technic].

A sequential microbiological control was carried out, using a total filtration method, on a sample selected at random from among those prepared on a daily basis and kept at room temperature for twenty-four hours, in order to determine any possible contamination by fungi, bacteria or yeasts. The results obtained were submitted to accumulated sum statistical sampling, the adjustment for which followed a binomial distribution model. For alpha = 0.05, levels of 0.04 and 0.12 respectively were fixed for acceptable quality (NCA) and for rejection (NCR). It was shown that the regulations on work in the unit for the preparation of NPT mixtures is satisfactory, as the latest results obtained are within the NCA established and, as a value, it is sufficiently exacting. The imperative need for routine microbiological control is also made clear, in order to maintain and improve the standard of quality in the section.


Cytogenetical and molecularbiological studies on a bovine XY female.

A bovine XY female in Holstein-Friesian heifer, which appeared as female with uterus and ovaries but did not show the estrus until 23 months old after the birth, was cytogenetically and molecularbiologically examined. As results of chromosome analysis, leucocyte and fibroblasts from skin, spleen and kidney examined had only metaphase plates with 60, XY. From these results and the clinical characteristics, this case was clearly diagnosed as a pure XY female. It was ascertained that the two genes, ZFY and AMG gene which located on the short arm of the Y chromosome (Yp) were detected in normal bulls and a XY female, but were not detected in normal cow, mother cow and half-sib heifer by Southern blotting.


[The correction of metabolic disorders in severely burned patients by enteral hyperalimentation].

An investigation was carried out of the metabolic processes, and some procedures for standardizing them, for patients with severe burns receiving uniformly distributed dosified high-calorie catheter alimentation, i.e. enteral hyperalimentation, in addition to the hospital's daily diet. Fifteen types of mixtures of Combustal were used, made and preserved ad hoc, and two commercial probe alimentation liquid products--Biosorbin-MCT (Pfrimmer-Kabi) and Fresubin (Fresenius AG). The average period taken to normalize the nitrogen balance was sixteen days counted from commencement of hyperalimentation. While it shifted the nitrogen balance figures from negative to positive, it was also seen to reduce A and C phospholipase activities in serum, while the level of excretion of nitrogenated amino acids and creatine remained high. During this time, pseudocholinesterase activity dropped, with the concentration of fibronectine in serum, which indicates low levels of biosynthetic processes and insufficiency in the reticuloendothelial system. The average value for the determination of lipids in general remained normal throughout the catheter feeding period. To ensure complete normalization of the metabolic process in patients suffering severe burns, enteral hyperalimentation must be extended for at least one month.


Anti-erythrocyte membrane antibodies detected in sera of dogs naturally infected with Babesia gibsoni.

Due to the potential for anti-erythrocyte membrane antibodies as possible enhancers of erythrocyte destruction, the presence of serum anti-erythrocyte membrane antibodies in 31 dogs with Babesia gibsoni infection admitted to a veterinary hospital was investigated by an enzyme linked immunosorbent assay (ELISA) and immunoblotting analyses. This infection resulted in an increase of anti-erythrocyte membrane antibodies in 84% (IgG) and 74% (IgM) of 31 infected dogs, respectively. This was confirmed by the similarity in the protein profiles of the erythrocyte membrane antigens immunoblotted with rabbit antiserum to dog erythrocyte membrane antigens and infected dog serum. These results suggest the production of anti-erythrocyte membrane antibodies was induced by B. gibsoni infection.


[Linoleic acid and the immune system. Controversies about lipid emulsions].

The selection of a given lipidic function for nutritional backup requires not only knowledge of the metabolism of the different existing lipidic emulsions and of their specific therapeutic indications, but also of their contraindications and controversies because, apart from their calorific value, the contribution of liposoluble vitamins and their function in preventing essential fatty acid deficiencies, we know that they are powerful metabolic modulators. This in associated with the fact that manipulation of dietary lipids (enteral or parenteral) can affect and modulate the response to the disease, attack or infection by improving or impairing the different immune functions. This review is focused on the scientific publications which have examined the varying effects of lipidic emulsions, in quantity and in quality (particularly linoleic acid) on the immune system, on the fatty acid composition of the cellular membranes and on the production of and prostaglandins and leukotrienes. An update is given of the known interrelation between lipids and immunity, with appraisal of triglycerides and long-medium -- and short-chain fatty acids, mixtures of medium -- and long-chain triglycerides, the proportions between infinity-3/infinity-6, and structured lipids.


Glomerular lesions in unilateral nephrectomized and diabetic (UN-D) mice.

Experimental diabetes was induced in both control and unilaterally nephrectomized male mice by injecting streptozotocin (SZ) (50 mg/kg x 5 days) one week after nephrectomy. The time course changes in the glomerular lesions were examined for up to 12 weeks after completion of the SZ-injection (12WAI). In unilateral nephrectomized and diabetic mice, mild segmental expansion of the mesangial area developed at 4WAI, and it progressed to prominent segmental glomerulosclerosis at 12WAI. In the electron microscopic examination at 12WAI, marked expansion of the mesangial area, segmental thickening of the glomerular basement membrane, fusion of the foot processes of podocytes and a prominent increase in the number of microvilli of capillary endothelial cells were observed. On the other hand, mild to moderate expansion of the glomerular mesangial area was only sporadically found in unnephrectomized diabetic mice at 12WAI. Interestingly, Bowman's capsules of diabetic mice were generally lined with flattened epithelia but those of non-diabetic mice with cuboidal or low columnar epithelia.


[Food composition tables: a practical appraisal].

We have revised the most common food composition tables published in Spain, France, Germany, England and the United States of America. In this article we have collected information concerning the points that may describe the size, format, numbers of items and numbers of nutrients in each table, so it can help professionals to know which of them may suit his needs properly.


[A histopathological study of the hepatic changes induced by parenteral nutrition in the rat].

Total parenteral nutrition is associated with hepatic metabolic and morphologic changes. Histological alterations induced by administration of glucose plus amino acids (group A) or glucose plus amino acids and lipids (group B) have been investigated in the rat. Parenteral nutrition provided 187 kcal/kg/day in both groups of animals. Rats in group B received 30% of nonprotein calories as lipids (Intralipid). In group A the most frequent histopathological findings were inflammation, portal edema and microesteatosis. Those alterations did not disappear by lipid addition, being frequent the existence of congestive phenomenons and esteatosis together with occasional pigment deposits in Kupffer cells. Those data suggest that supplementation of solutions with LCT-base lipid emulsions does not compensate for the histopathological alterations induced by parenteral nutrition.


Serum resistance and aerobactin iron uptake in avian Escherichia coli mediated by conjugative 100-megadalton plasmid.

A total of 115 strains of Escherichia coli isolated from chickens with colisepticemia in Japan were examined for chicken lethality and virulence factors. It was found that serum resistance and aerobactin-mediated iron uptake are the most prevalent characteristics in these strains. Among them, S-20, a representative virulent strain of serotype O2, was further studied. S-20 harbored a conjugative 100-megadalton (Mdal) plasmid, designated pKI100. Curing and reintroduction experiments showed that pKI100 encodes both serum resistance and aerobactin-mediated iron uptake, and the diminished virulence of the pKI100-cured strain was fully restored by the reintroduction of the plasmid. These results demonstrated that pKI100 is the virulence plasmid of the S-20 strain, and that serum resistance and aerobactin-mediated iron uptake are the virulence factors in E. coli strains which cause avian colibacillosis.


Developmental capacity of reconstituted mouse embryos: influences of nucleus and cytoplasm sources.

This study was undertaken to examine the developmental capacity of reconstituted mouse embryos, and the influences of nucleus and cytoplasm on the development of these embryos following reciprocal pronuclear transplantation between in vitro 2-cell blocked and nonblocked embryos. Karyoplast containing pronuclei was transferred into the perivitelline space of the enucleated zygote and fused to cytoplasm with electrofusion. Maximum fusion rate was obtained when a field strength of 1.5 kV/cm was used. The fusion rates were high (86.2 +/- 3.2 to 90.6 +/- 2.0%) regardless of the strains of donor nucleus and recipient cytoplasm. Developmental rates of reconstituted embryos to the blastocyst stage, which were similar to that of the F1 (C57BL/6J x CBA) control were high when F1 embryos were used as the cytoplasm recipients (88.8 +/- 1.5 and 91.9 +/- 2.0%). When ICR embryos were used as the recipient cytoplasm, developmental rates were significantly reduced (71.5 +/- 2.9 and 54.1 +/- 3.2%), and affected by the source of nucleus. There were no significant differences in the cell number of embryos that developed to blastocysts and in the developmental rates to live young among the embryos reconstituted with different nuclei and cytoplasm, and the ICR control. The results of this study show that the development of reconstituted embryos is hardly affected by nuclear transplantation and electrofusion procedures. It is indicated that the recipient cytoplasm, rather than the donor nucleus, has the greater influence on the in vitro development of the reconstituted embryos to the blastocyst stage.


Interaction between Bordetella bronchiseptica and toxigenic Pasteurella multocida on the nasal mucosa of SPF piglets.

The interaction between Bordetella bronchiseptica and type D toxigenic Pasteurella multocida was studied in five groups of 4 specific-pathogen-free (SPF) piglets each. At 28 days of age, piglets of groups 3 and 4 were inoculated into both nostrils with 10(8) colony-forming-units (CFU) of a non-dermonecrotic toxin (DNT)-producing, phase I strain of B. bronchiseptica. Piglets of groups 1 and 3 were treated intranasally with a sonic extract of the non-toxic strain of B. bronchiseptica and those of groups 2 and 4 with B. bronchiseptica DNT into the left nostril. Sonic extract and DNT treatment was started at 33 days of age and lasted for 5 days. Piglets of group 5 served as controls. At the age of 37 days, piglets of all groups except group 5 were inoculated into both nostrils with 5 x 10(7) CFU of toxigenic P. multocida. At slaughter at 50 days of age, P. multocida was recovered from the left nasal cavity of 3 piglets of group 2 and all piglets of group 4. In piglets inoculated with B. bronchiseptica DNT the mucosal epithelial cells of the left nasal cavity showed loss of cilia, regressive lesions such as vacuolation, karyopycnosis and necrosis, hypertrophy of the epithelium, infiltration of the epithelium and submucosa by inflammatory cells, could also be seen. The results suggest that action of the B. bronchiseptica DNT on the nasal mucosa is a precondition of the growth of P. multocida in the nasal cavity.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of estrogen and progesterone on the development of the mammary gland and the associated blood vessels in ovariectomized mice.

Effects of estrogen and progesterone on the first abdomino-inguinal mammary gland and the associated blood vessels in ovariectomized mice were investigated morphometrically, and light and electron microscopically. Although there were no significant differences in the area of the fat pad of the mammary gland among the experimental groups, the area of the mammary parenchyma and diameters of the blood vessels supplying the mammary gland, i.e., A. et V. circumflexa ilium profunda and A. et V. epigastrica caudalis superficialis, reached the maximal value in ovariectomized mice treated with estradiol (E) + progesterone (P). Similarly, the blood capillaries around buds and ducts of the mammary gland were most densely distributed in E + P treated mice. In the adipose tissues of the mammary stroma in intact mice, fat cells were of multilocular type in the peripheral regions around the main vessel, and of unilocular type in the other part. In E and E + P treated mice, however, fat cells were mostly of unilocular type. These findings suggest that the formation of mammary fat tissues may occur in advance of that of the mammary parenchyma. By TEM, epithelial cells of the mammary parenchyma contained a large number of mitochondria and ribosomes, well-developed Golgi apparatus and rER, and large lipid droplets. Endothelial cells of blood capillaries displayed numerous pinocytotic vesicles, longer marginal folds and microvillous processes. Each organelle in these two cell types increased in number or developed to a greater degree in E + P treated mice than the other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Interrelationship between lipid droplets and mitochondria in brown adipocytes of the hamster.

The interrelationship between lipid droplets and mitochondria in the interscapular brown adipose tissue of the hamster was investigated by electron microscopy. The membranous structure of mitochondria began to degenerate at the site in contact with lipid droplets. From that site, mitochondrial cristae also began to collapse. After being completely surrounded by lipid droplets, these degenerating mitochondria became vacuolated within them. Finally, lipid infiltrated into these vacuoles. Mitochondrion-like structures found in the lipid droplets may be possibly residues of the degenerating mitochondrial membranes.


Sudden cardiac death in calves with experimental heavy infection of Strongyloides papillosus.

For obtaining the preliminary data on the pathogenesis of sudden death in calves naturally heavily infected with Strongyloides papillosus, we monitored 8 Holstein calves experimentally infected with the larvae on electrocardiographic and pneumographic changes. Six calves died suddenly on days 11 to 17 after infection. Sinus tachycardia had been recorded continuously since 1 to 6 days before death. Heart rates increased gradually until death. Since 1 or 2 days before death, various patterns of tachyarrhythmia and bradyarrhythmia had been observed among patterns of sinus tachycardia. Arrhythmias included serious ventricular premature beat, paroxysmal ventricular tachycardia, complete atrioventricular block and so on. The terminal pattern observed suddenly in all of the cases was ventricular arrhythmias consisting of serial ventricular tachycardia, flutter and fibrillation, which were followed by respiratory arrest. Abnormal pneumograms were not obtained before the terminal ventricular fibrillation. Two of 8 calves recovered from the infection, only one of which showed sinus arrest and the second degree of atrioventricular block transiently. We concluded that calves heavily infected with the larvae died due to sudden cardiac arrest.


Immunohistochemical morphometry of pancreatic islets in the cat.

The application of immunohistochemical technique with antisera for glucagon (Glu), insulin (Ins), somatostatin (Som) and pancreatic polypeptide (PP) to serial sections of the cat pancreas permitted the quantitative evaluation of the population of 4 endocrine cell types and that of the area, larger diameter and density of islets. The pancreas was divided macroscopically into the 4 portions, duodenal, gastric, anastomotic and splenic. The duodenal portion was characterized by the localization of PP-immunoreactive (IR) cell-rich islets, the dissemination of PP-IR cells in the exocrine parenchyma and the absence of Glu-IR cells. In the duodenal portion, the area, the larger diameter and the density of islets were significantly smaller than those in the other 3 portions. On the contrary, the other 3 portions were marked with the deficiency of PP-IR cells and the existence of Glu-IR cell-rich islets. Ins-IR cells, identified as compact cell masses without any other types of cells, occupied a major part of every islet, composing much the same population throughout the 4 portions. The Som-IR cell population appeared to be closely in parallel with the Glu-IR cell population in all of the portions. It is concluded that all islets are similar in the Ins-IR cell population, but different in the complementary arrangement of Glu- and PP-IR cells. Based on this difference, 2 types of islets can be classified.


Effect of 50 kilobase-plasmid, pKDSC50, of Salmonella choleraesuis RF-1 strain on pig septicemia.

Salmonella choleraesuis strains with and without 50-kilobase plasmid (pKDSC50) were intravenously inoculated into Yorkshire pigs. By the inoculation of 7.2 x 10(5) - 3.5 x 10(7) cells, RF-1 strain with pKDSC50, but not 31N-1 strain without the plasmid, caused a septicemia. The inoculation of 8.7 x 10(9) RF-1 cells killed pigs at 2-4 day postinfection with severe hemorrhage on the whole body. Pigs with a similar number of 31N-1 cells (8.3 x 10(9) cells), showed milder hemorrhage, and they died at 6 day postinfection. These results indicated that pKDSC50 is required for RF-1 strain to express the full virulence causing a heavy cutaneous pig septicemia.


Growth activity of bovid herpesvirus 1 in bovine follicular oocytes with cumulus cells.

Bovine follicular oocytes collected from bovine ovaries were exposed to bovid herpesvirus 1 (BHV-1). After washings, these oocytes were cultured to mature. As a result BHV-1 could not be removed from the oocytes and could replicate in the oocytes with cumulus cells, but not in the oocytes without the cells. Moreover, the specific fluorescence for BHV-1 was detected in the cumulus cells by a indirect immunofluorescent technique. Therefore these findings suggested BHV-1 could be absorbed in the oocytes but the replication of BHV-1 was done in the cumulus cells.


Enhancing effect of butorphanol on medetomidine-induced sedation in pigs.

Enhancing effect of an opiate agonist-antagonist butorphanol (0.2 mg/kg) on sedation induced by medetomidine (80 micrograms/kg) was evaluated in pigs. Butorphanol significantly enhanced the depth of medetomidine-induced sedation and prolonged the duration of that assessed by posture score and spontaneous movement of pigs. The combination of medetomidine and butorphanol produced excellent muscle relaxation and moderate surface analgesia which was enough for procedures with mild pain in pigs.


Species-specific kinetics of mouse renin contribute to maintenance of normal blood pressure in transgenic mice with overexpressed human angiotensinogen.

Angiotensinogen, the precursor of a vasoactive octapeptide angiotensin II, is the only known natural substrate of renin, and its reaction exhibits strict species specificity and is the rate-limiting step in the renin-angiotensin system that controls the blood pressure. We measured blood pressure and heart rate of the transgenic mice with overproduced human angiotensinogen, and showed no significant difference in these parameters between transgenic and nontransgenic mice. We also provided evidence that mouse renin could not cleave human angiotensinogen, indicating a lack of angiotensin production from the human substrate. These results suggested that the blood pressure of transgenic mice is normally maintained, probably due to the inability of mouse renin to release angiotensin from the transgene products.


Studies on closure of the ductus arteriosus in perinatal rats.

Measurements of the inner diameters (calibers) of the ductus arteriosus (DA) and pulmonary artery (PA) were made in late fetal rats and newborn rats, the latter being obtained by spontaneous or caesarean delivery. The fetal and newborn pups were frozen instantly with an acetone-dry ice mixture. The chests of these whole-body frozen pups were shaved with a surgical knife gradually from the back toward the ventral side to expose the DA and PA for measurements of their calibers. As a result, it was revealed that the DA was almost closed 180 min after birth, but that the closure and shrinkage of the DA were accelerated to some extent by caesarean delivery. On the other hand, there was no remarkable change in the PA throughout the postnatal period observed, regardless of the type of delivery, spontaneous or caesarean.


Implantation in both of the uterine horns after the unilateral intrauterine insemination in rabbits.

Artificial insemination was carried out by injecting semen into the upper part of the left uterine horn in rabbits with a duplex uterus. Ovulation was then induced by administration of hCG. As a result, implanted fetuses were observed not only in the left uterine horn but also in the right uterine horn. However, when similar insemination was carried out after ligation of the right utero-tubal junction, fertilization did not occur in the right oviduct. From these finding, we conclude that some of the spermatozoa injected into the left uterine horn was discharged into the vagina, entered the right uterine cervical canal, and reached the right oviduct to fertilize the ova.


Pituitary dependent hyperadrenocorticism in a cat.

A cat that was suspected some insulin resistance was diagnosed as pituitary dependent hyperadrenocorticism from an adrenocorticotropic hormone (ACTH) stimulation test, dexamethasone suppression test and measure of endogenous plasma ACTH concentration. Histopathological examination revealed chromophobe adenoma in pituitary gland and hyperplasia in adrenal cortex.


Listeric septicemia with meningitis in a neonatal calf.

A 9-days-old calf which had exhibited depression and difficulty to stand and walk was examined pathologically and bacteriologically. The primary pathological changes consisted of multifocal necrosis in several visceral organs and fibrinopurulent meningitis. The necrotic lesions were most frequently found in the liver, and accompanied with mononuclear cell infiltration and Gram-positive small bacilli. The organisms were also present in the foci of mononuclear cells at the central gray matter of the mesencephalon. Listeria monocytogenes was isolated from the brain and other organs of the whole body.


Cardiopathological observation on a case of persistent ventricular tachycardia in a pony mare.

A 20-year-old pony mare with persistent ventricular tachycardia (VT) was examined cardiopathologically. At necropsy, the heart was enlarged and rounded with both ventricular dilatation. In a longitudinal section of the ventricular septum, a large grayish white patchy lesion (5 x 25 mm) was detected in the relatively higher portion. Microscopically, the lesion was extensive myocardial fibrosis located in the vicinity of the proximal part of the left bundle branch. Partially the fibrotic lesion was in contact with the branch. Such a lesion might play an important role in creating a suitable background for the development of VT via automaticity or reentry mechanism.


Changes in the level of plasma cortisol, progesterone and total testosterone in developing hairless dogs.

Changes in the levels of plasma cortisol, progesterone and total testosterone were examined in developing hairless and haired dogs. Cortisol levels in the hairless dogs seemed to be higher than those in haired dogs within the age of 4-5 weeks. No apparent changes were seen in the level of plasma progesterone between the groups of hairless females and haired females. Total testosterone levels in hairless males showed to be significantly lower than those in haired males at the age of 13-21 weeks.


Chromosome-sized DNA of Malassezia pachydermatis by pulsed-field gel electrophoresis.

The genome of Malassezia pachydermatis isolates from dogs was resolved into six chromosomes by using pulsed-field gel electrophoresis and their molecular sizes were calculated as 820, 1,100, 1,400, 1,470, 1,660 and 1,820 Kb, respectively. Comparison of electrophoretic patterns suggested that the chromosomes of M. pachydermatis were homozygous.


Changes in anti-erythrocyte membrane antibody level of dogs experimentally infected with Babesia gibsoni.

To account for the conflict between the excessive destruction of erythrocytes and the number of parasitized erythrocytes in dogs infected with Babesia gibsoni, we examined the correlation between anti-erythrocyte membrane antibody level (AEMAL) and the number of erythrocytes (RBC count) in dogs with experimentally induced babesiosis using hematological examination and an enzyme linked immunosorbent assay (ELISA). In the infected dogs without splenectomy, more prominent reduction in RBC count accompanied with the elevated AEMAL was presented than anticipated from parasitemia until the 21st day. Furthermore, autoagglutinated erythrocytes and spherocytes were demonstrated in blood films. These results suggest that a humoral immunologic mechanism may be involved in a decrease in RBC count in dogs infected with B. gibsoni.


The first outbreak of fowl cholera in Muscovy ducks (Cairina moschata) in Japan.

The first outbreak of fowl cholera occurred in a flock of Muscovy ducks (Cairina moschata) in Okinawa Prefecture of Japan in November 1990. Fifty (25%) of 200 birds in a farm died of an acute disease. Remaining birds recovered after treatment with oxytetracycline. Pasteurella multocida subsp. multocida was isolated in pure culture from all tissues tested from two dead birds. Serovars of the isolates were identified as Carter's capsular type A. Heddleston's type 3.4.12, and Namioka's type 5:A which have not been demonstrated in Japan. Pathologically, multiple necrosis and bacterial aggregates were prominent in several organs, particularly in the liver. The isolate killed chickens when inoculated intravenously at a concentration of 10(8) colony forming units.


A scanning electron microscopic study on the difference between white and brown adipose tissues in the rat.

Lipid globules in the white and brown adipose tissues in the rat were well preserved with prolonged postfixation in OSO4. The difference between the surface of white adipose tissue and that of brown adipose tissue was observed in such a way that the surface of the brown adipocyte was lumpy owing to the presence of intracellular lipid globules, and that the surface of the white adipocyte was considerably smooth. After the matrix of lipid globules was dissolved during specimen preparation, the subgranules embedded in the matrix were disclosed on the inner surface of the lipid globules. The cut surface of the white adipocyte contained a large lipid droplet and the amount of cytoplasm was poor, whereas the brown adipocyte had several lipid droplets and many small granules in the cytoplasm.


Biochemical parameters following capture myopathy in one Arabian oryx (Oryx leucoryx).

Twenty-three biochemistry parameters and hematocrit were followed during 10 days in a 13 months old Arabian Oryx (Oryx leucoryx) during capture myopathy. An increase was found in bilirubin, creatine-kinase, alanine aminotransferase, and aspartate aminotransferase levels, but not in potassium level. Most of the parameters analyzed were the first given for this species.


Production of granulocyte/macrophage colony-stimulating factor in human airways during allergen-induced late-phase reactions in atopic subjects.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) are hematopoietic growth factors that have been shown to induce proliferation and activation of inflammatory cells, and may play a role in allergic reactions. Since little is known about the involvement of cytokines in allergic inflammation in the lung, the levels of GM-CSF and IL-3 were measured in bronchoalveolar lavage (BAL) fluids obtained in the late phase after segmental lung antigen (Ag) challenge in 14 allergic rhinitis subjects with or without bronchial asthma. BAL fluids either after Ag (ragweed, dust mite, or timothy) or saline control challenge were recovered 19 h later. In 6 of the 14 patients, BAL fluids were concentration-dialyzed (20x) and assayed for cytokine activity. Cytokine assays were performed using the human megakaryocytic leukemic cell line M-07e, which is responsive to either GM-CSF or IL-3. The level of GM-CSF-equivalents was approximately 25 times higher in Ag-challenged sites (49.9 +/- 12.7 pg/ml; mean +/- SEM), compared to saline challenge sites (2.2 +/- 1.0, p < 0.01, n = 9). Neutralization experiments using a polyclonal specific antibody (Ab) against GM-CSF and IL-3 revealed that the bulk of the activity was GM-CSF. BAL fluids from Ag- and saline-challenged sites in one nonatopic subject contained no significant GM-CSF activity. Furthermore, the level of GM-CSF in Ag-challenged BAL fluid and the percentage of eosinophils in BAL from each subject correlated significantly (r = 0.73, p < 0.005, n = 14).(ABSTRACT TRUNCATED AT 250 WORDS)


Modulation of blood-brain barrier permeability by tumor necrosis factor and antibody to tumor necrosis factor in the rat.

In an attempt to understand the role of TNF in the central nervous system (CNS) pathophysiologic events associated with bacterial meningitis, we examined the effect of intravenous vs. intracisternal administration of TNF alpha on penetration of circulating 125I-labeled albumin into cerebrospinal fluid (CSF) and CSF white blood cell (WBC) counts in rats. Intracisternal administration of tumor necrosis factor alpha (TNF-alpha) resulted in dose- and time-dependent alterations of the CSF penetration and CSF WBCs, while intravenous administration of TNF-alpha did not induce any changes. These changes by intracisternal TNF were abolished by heat treatment of TNF or coadministration of MAb to TNF-alpha. Mab to TNF-alpha also significantly reduced the CSF penetration of circulating albumin in experimental hematogenous Haemophilus influenzae type b meningitis in infant rats but this salutary effect required both intravenous and intracisternal administration. However, MAb to TNF-alpha failed to affect CSF pleocytosis in experimental hematogenous meningitis. These findings suggest that some of CNS pathophysiologic changes in bacterial meningitis may be a result of the local production of TNF but other host inflammatory responses may also participate in CNS inflammation in hematogenous bacterial meningitis.


Transforming growth factor-beta inhibits human T-cell proliferation through multiple targets.

This study investigates further the inhibitory effects of transforming growth factor-beta (TGF-beta) on human T-lymphocyte responses to mitogenic stimulation. T cells were stimulated either with mitogenic concentrations of PHA or with submitogenic concentrations of Con A followed by the addition of IL-2. DNA synthesis ([3H]thymidine incorporation) in both systems was inhibited by 60-69% in the presence of TGF-beta, with maximal reduction occurring on days 4 and 5 of culture. Cell surface expression of transferrin receptor (TfR) and IL-2 receptor-alpha (p55) were inhibited by 20-80% in the Con A/rIL-2 system and 20-45% in the PHA system in the presence of TGF-beta. In addition, mitogen-induced up-regulation of TfR and IL-2R mRNA levels were inhibited by TGF-beta. Finally, we investigated the effect of TGF-beta on the assembly of clathrin monomers into assembled coated pits and vesicles, and essential step in TfR and IL-2R alpha turnover. Stimulation of T cells using either mitogen system resulted in an increase in the level of assembled clathrin, which was almost completely inhibited by TGF-beta. These findings suggest that TGF-beta may act at several sites in mitogen-mediated proliferative pathways to contribute to the inhibition of T-cell proliferation.


Human hybridoma-derived suppressor factor 160 and transforming growth factor-beta are different molecules.

We have previously identified a suppressor factor (SF), designated 160 constitutively produced by human T-T-cell hybridomas generated by fusing Con A-activated human peripheral blood lymphocytes from a normal donor with cells of the Jurkat tumor T-cell line (Hybridoma 8:127-151, 1989). The 160 SF inhibited in vitro proliferative responses to polyclonal activators and allogeneic cells, and immunoglobulin synthesis and secretion of human and mouse lymphocytes. We investigated whether the hybridoma-derived 160 SF and transforming growth factor-beta (TGF-beta) are distinct molecules. TGF-beta has been shown to inhibit a number of lymphocyte responses. In agreement with our previous findings, the 160 SF abrogated the proliferative responses of human peripheral blood mononuclear cells (PBMC) to mitogens and allogeneic cells in mixed lymphocyte culture. In contrast, TGF-beta, added to the PBMC cultures at the same time with the mitogen or the stimulating allogeneic cells, had no effect on the proliferative response. Acid treatment of the 160 SF completely abolished the 160 SF activity. In contrast, this treatment results in activation of the latent TGF-beta form to the active form, and acidification does not affect the function of existing active TGF-beta. A polyclonal anti-TGF-beta antibody did not detect TGF-beta by Western blotting in concentrated (10x) 160 SF preparations. In addition, the 160 SF did not induce the anchorage-independent growth of NRK fibroblasts in the presence of EGF.TGF-beta at concentrations as low as 1 ng/ml, in the presence of EGF, induced the anchorage-independent growth of the anchorage-dependent indicator NRK cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Contrasting effects of interferon-gamma and interleukin-4 on neopterin generation from human adherent monocytes.

The production of neopterin is closely correlated with activation of cell-mediated immunity. Neopterin appears to be produced by human macrophages specifically stimulated with interferon-gamma (IFN-gamma). Interleukin-4 (IL-4), a B and T stimulatory factor, has recently been shown to inhibit monocyte/macrophage functions, including the ability to suppress monocyte-generated cytokines. In this report we confirmed previous studies that identified the monocyte/macrophage as the main producing cell among human blood cells and that secretion is stimulated by IFN-gamma and lipopolysaccharides (LPS). IL-4 inhibits the generation of neopterin from unstimulated monocytes. This inhibitory effect was dose dependent and occurred at concentrations lower than 0.01 ng/ml. However, IL-4 had only a minimal inhibitory effect on LPS-induced generation of neopterin and could not reverse IFN-gamma-induced neopterin secretion from adherent monocytes. Furthermore, we report that LPS induced IFN-gamma production in monocyte culture. This production is strongly inhibited by IL-4 treatment. These findings indicate that IL-4 can regulate the synthesis of neopterin by adherent blood mononuclear cells and provide further evidence that LPS-induced neopterin in macrophages may act by IFN-gamma-independent mechanisms.


Beta-endorphin inhibits interleukin-2 release and expression of interleukin-2 receptors in concanavalin A-stimulated splenic lymphocytes.

beta-endorphin, when added at the same time as the mitogenic lectin concanavalin A to mouse BALB/c spleen lymphocytes, inhibits cell proliferation. The suppressive effect of beta-endorphin is not exercised through a cAMP-dependent mechanism and is also observed when splenic lymphocytes are stimulated with phytohemagglutinin (4 micrograms/ml), anti-CD3 monoclonal antibody, or the Ca2+ ionophore A23187 (250 nM) and phorbol 12-myristate 13-acetate (1 ng/ml). The inhibitory effect of beta-endorphin on lymphocyte proliferation is dose and time dependent: when beta-endorphin is added 20 h after Con A stimulation no suppression of lymphocyte proliferation is observed. beta-Endorphin inhibits, in a dose-dependent manner, the release of interleukin-2 in concanavalin A-stimulated splenic lymphocytes, measured 24 h after stimulation. beta-Endorphin also controls the appearance of interleukin-2 receptors in the plasma membrane, but does not regulate the expression of the c-myc protooncogene. These data indicate that beta-endorphin inhibits lymphocyte activation signal transmission, downstream the generation of the second messengers Ca2+ and diacylglycerol and the expression of the protooncogene c-myc, by blocking interleukin-2 release and interleukin-2 receptors expression. Once the cells are in the G1 stage, beta-endorphin is no longer able to block lymphocyte proliferation.