Linkage study in a large pedigree with Stickler syndrome: exclusion of COL2A1 as the mutant gene. A three generation family with Stickler syndrome is reported. Affected patients exhibited myopia with frequent retinal detachment or glaucoma. Most of them had characteristic facial dysmorphism, the Pierre-Robin sequence being observed in four individuals. Neonatal radiological signs of the Weissenbacher-Zweymüller syndrome were also noticed but early arthopathy was not reported in adults. Restriction fragment length polymorphism studies with the type II collagen gene (COL2A1) showed a recombination event between the disease locus and COL2A1, thus excluding collagen type II as the candidate gene. Although the calculation of the likelihood of genetic heterogeneity versus homogeneity based on 10 families was not statistically significant, we suggest that a second locus is probably involved in this highly variable syndrome.


Somatic cell mapping, polymorphism, and linkage analysis of bovine prolactin-related proteins and placental lactogen. The bovine prolactin gene family includes novel members expressed in the fetal placenta that are distinct from placental lactogen. In this study, we investigated the genetic organization of four members of this gene family (PRP1, PRP3, PRP6, and PRP10) as well as placental lactogen (PL). Using a bovine-rodent hybrid somatic cell panel, all five genes were assigned to bovine chromosome 23, which contains prolactin and the major histocompatibility group (BOLA). Restriction fragment length polymorphisms were detected by all probes in breeding populations with the restriction enzyme MspI, whereas no polymorphisms were detected with BamHI. EcoRI, HindIII, TaqI, and PstI produced polymorphic fragments with some but not all of the probes tested. A PRP10 polymorphism, which is apparently the result of a insertion/deletion event, detected polymorphism frequency differences between Bos indicus and Bos taurus. No recombinational events were observed with these probes and prolactin using linkage analysis involving 91 American Holsteins. The bovine prolactin gene family was incorporated into a linkage group containing CYP21. Our studies demonstrate that members of the bovine prolactin gene family have a close physical association with each other, and all members demonstrate genetic variability in the breeding population.


Molecular and genetic characterization and physical mapping of 11 new markers detecting multiallele restriction fragment length polymorphisms on the short arm of human chromosome 3. Genetic markers with high degrees of polymorphisms are of vital importance in the construction of high resolution (2-4 cM) linkage maps of human chromosomes as specified in the short-term goals of the Human Genome Initiative. In this paper, we report on molecular and genetic characterization and physical localization of 11 new multiallele restriction fragment length polymorphism markers on human chromosome 3p. Ten of these represent three- and four-allele polymorphisms of the base substitution type probably at two adjacent restriction sites. One has been identified as a novel mini-satellite sequence comprising a variable copy number tandem repeat array of a G/T-rich 79-bp sequence. This collection of multiallele polymorphic (PIC values: 0.40-0.60) markers should prove valuable and increase the resolution power of the available chromosome 3p genetic markers.


Cloning of the human cholesterol 7 alpha-hydroxylase gene (CYP7) and localization to chromosome 8q11-q12. Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is a microsomal cytochrome P450 that catalyzes the first step in bile acid synthesis. In this paper, we describe the cloning, characterization, and chromosomal mapping of the human 7 alpha-hydroxylase gene (CYP7). The gene spans 10 kb and contains six exons and five introns. The exon-intron boundaries are completely conserved between the human and rat genes. Sequencing of the 5' flanking region revealed consensus recognition sequences for a number of liver-specific transcription factors. The human CYP7 gene was mapped to chromosome 8q11-q12 using both mouse-human somatic cell hybrids and in situ chromosomal hybridization studies. A total of four single-stranded conformation-dependent DNA polymorphisms and an Alu sequence-related polymorphism were identified. Of the individuals analyzed, 80% were heterozygous for at least one of these five polymorphisms. The localization and characterization of the human 7 alpha-hydroxylase gene, as well as the identification of polymorphisms, provide the molecular tools necessary to investigate the role of this gene in disorders of cholesterol and bile acid metabolism.


Analysis of the 5' flanking sequence of the G gamma globin gene by denaturing gradient gel electrophoresis confirms the heterogeneity of the Bantu beta S haplotype. The GC-->TT polymorphism recently described at positions -1106 and -1105 in the 5' flanking region of the G gamma globin gene for the Bantu beta S haplotype was analysed by denaturing gradient gel electrophoresis. We studied 108 beta S chromosome and 122 beta A chromosomes. The TT sequence was found as follows: in all of 80 chromosomes bearing the Bantu beta S haplotype with the 6-bp deletion -400 nt from the G gamma gene in 3 out of 5 Bantu beta S chromosomes without the deletion, in 1 out of 122 beta A chromosomes from different ethnic origins but in none of 23 beta S chromosomes bearing the Senegal, Benin or Cameroon haplotypes. These results confirm the heterogeneity of the Bantu beta S haplotype and allow a tentative evolutionary sequence for the different alleles at this locus to be presented.


Generation of 19 STS markers that can be anchored at specific sites on human chromosome 21. Sequence-tagged sites (STSs) are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR) and can be used to construct long-range physical maps of chromosomal DNA. These STSs can be detected by PCR assays developed by reference to data obtained from the sequencing of restriction fragment length polymorphism-DNA markers for chromosome 21, which were derived from recombinant lamba-phage and plasmid clones made from DNA of a human-hamster hybrid cell line. In this report, we describe the generation of 19 new STSs that are specific for human chromosome 21.


Localization of insulin-2 (Ins-2) and the obesity mutant tubby (tub) to distinct regions of mouse chromosome 7. A DNA mapping panel derived from an interspecific backcross was used to position the mouse insulin-2 locus (Ins-2) on Chromosome 7, near H19 (0/114 recombinants) and Th (1/114 recombinants). Ins-2 is part of a human-mouse conserved linkage group that includes Th, H19, and Igf-2. Analysis of segregation in the F2 generation from the cross C57BL/6J-tub/tub x CAST/Ei demonstrated that Ins-2 and the obesity mutant tubby (tub) are distinct loci, thus eliminating Ins-2 as a candidate gene for tub. These results also refine the estimated genetic distance between tub and Hbb to 2.4 +/- 1.4 cM. The predicted location for a human homolog of tubby is HSA 11p15.


A linkage map of mouse chromosome 19: definition of comparative mapping relationships with human chromosomes 10 and 11 including the MEN1 locus. A linkage map of mouse Chromosome (Chr) 19 was constructed using an interspecific cross and markers defined by restriction fragment length variants. The map includes 20 markers, 9 of which had not been mapped previously in the mouse. The data further defined the relationship between genes on mouse Chr 19 and those on the long arm of human Chr 10 and the pericentric region of the long arm of human Chr 11. The comparative mapping analysis suggests that the proximal segment of mouse Chr 19 may contain the MEN1 locus and that the current study has identified additional genes that may be useful for positional cloning of this putative tumor suppressor gene.


Identification and mapping of six microdissected genomic DNA probes to the proximal region of mouse chromosome 1. Six independent DNA probes, lambda Mm1C-150, lambda Mm1C-153, lambda Mm1C-156, lambda Mm1C-162, lambda Mm1C-163, and lambda Mm1C-165, have been isolated from a library of microdissected fragments from mouse chromosome 1, spanning cytogenetic bands C2 to C5. These DNA probes have been mapped by restriction fragment length polymorphism analysis with respect to 12 marker loci previously assigned to this portion of mouse chromosome 1, in a panel of 251 segregating Mus spretus x C57BL/6J interspecific backcross mice. The gene order and intergene distances were determined by segregation analysis to be centromere- lambda Mm1C-162-11.1 cM-Col3a1-8.8 cM-Len-2-2.6 cM-lambda Mm1C-163-1.6 cM-Fn-1-1.6 cM-Tp-1-0.8 cM-lambda Mm1C-165/Vil-0.4 cM-Inha-2.8 cM-lambda Mm1C-153-2.4 cM-lambda Mm1C-156-1.2 cM-Pax-3-5.6 cM-Akp-3-0.8 cM-Acrg-2.0 cM-Sag-0.5 cM-Col6a3-1.8 cM-lambda Mm1C-150-15.4 cM-Ren1,2. Four of these probes map within a chromosome 1 segment that is homologous to human chromosome 2q. Southern blotting analyses indicate that one of these anonymous probes, lambda Mm1C-165, detects DNA fragments highly conserved across species. These novel polymorphic probes should prove useful for linkage and physical mapping of this chromosomal region.


Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6. The CYP2D gene cluster on human chromosome 22 containing the functional cytochrome P450 gene CYP2D6 and two or three highly homologous pseudogenes is involved in a clinically important variation in the inactivation of drugs and environmental chemicals. Several mutant haplotypes of CYP2D6 have been identified by restriction analysis and by PCR-based allele-specific amplification. To understand the evolutionary sequence of mutational events as well as recently discovered interracial differences, we analyzed the arrangement of the CYP2D haplotype containing a common mutant allele of CYP2D6 associated with a XbaI 44-kb fragment. This haplotype contains four CYP2D genes instead of three. Comparison of the sequences of these genes with those of previously characterized haplotypes suggests that an early point mutation was followed by a crossover and a gene conversion event, the latter found preferentially in Caucasians. These data are consistent with the rapid evolution of this locus during "plant-animal warfare" with practical consequences for present-day defense of the organism against environmental adversity.


Assignment of genes encoding a unique cytokine (IL12) composed of two unrelated subunits to chromosomes 3 and 5. IL12 (formerly NKSF or CLMF) is a unique cytokine composed of two unrelated disulfide-linked subunits. The larger 40-kDa subunit (p40) is a member of the cytokine receptor family, and the smaller 35-kDa subunit (p35) is related to IL6 and GCSF. The chromosomal localization of these two subunits was determined by PCR analysis of DNA from rodent-human hybrids. More refined mapping was obtained by PCR analysis of hybrids containing translocation chromosomes and for p40, by analysis of radiation hybrids. The subunits map to different chromosomes: p40 (IL12B) to 5q31-q33 and p35 (IL12A) to 3p12-3q13.2.


Mapping of the regulatory subunits RI beta and RII beta of cAMP-dependent protein kinase genes on human chromosome 7. The genes encoding the regulatory subunits RI beta (locus PRKAR1B) and RII beta (locus PRKAR2B) of human cAMP-dependent protein kinase have been mapped in the basic CEPH (Centre d'Etude du Polymorphisme Humain) family panel of 40 families to chromosome 7p and 7q, respectively, using the enzymes HindIII and BanII recognizing the corresponding restriction fragment length polymorphisms (RFLPs). Previous data from the CEPH database and our present RFLP data were used to construct a six-point local framework map including PRKAR1B and a seven-point framework map including PRKAR2B. The analysis placed PRKAR1B as the most distal of the hitherto mapped 7p marker loci and resulted in an unequivocal order of pter-PRKAR1B-D7S21-D7S108-D7S17-D7S149- D7S62-cen, with a significantly higher rate of male than female recombination between PRKAR1B and D7S21. The 7q regulatory gene locus, PRKAR2B, could also be placed in an unambigous order with regard to the existing CEPH database 7q marker loci, the resulting order being cen-D7S371-(COL1A2,D7S79)-PRKAR2B-MET-D7S87++ +-TCRB-qter. Furthermore, in situ hybridization to metaphase chromosomes physically mapped PRKAR2B to band q22 on chromosome 7.


Methylation of the DXS255 hypervariable locus 5' CCGG site may be affected by factors other than X-chromosome activation status. Differences in the methylation status of certain cytosine residues between active and inactive X chromosomes can be used to determine X-inactivation in females heterozygous for X-linked restriction fragment length polymorphisms. We have studied methylation patterns in 105 females heterozygous at the DXS255 locus by Southern blotting of PstI and MspI or HpaII double digests and hybridization with the probe M27B. Unequivocal patterns of unilateral or bilateral X-inactivation were obtained in 15/64 and 49/64 cases, respectively. In the remaining 41 cases the results were unclear due to the absence of HpaII digestion of one or both PstI fragments. In 7 samples an unequivocal digestion pattern was demonstrated on repeat analysis, suggesting that the initial ambiguous pattern was due to incomplete HpaII digestion. In certain individuals, methylation at the 5' CCGG DXS255 locus may be affected by factors other than X-inactivation, making analysis of clonality with the M27B probe impossible. These individuals should be clearly identified in studies of clonality.


The accuracy of DNA sequences: estimating sequence quality. In this paper we describe a method for the statistical reconstruction of a large DNA sequence from a set of sequenced fragments. We assume that the fragments have been assembled and address the problem of determining the degree to which the reconstructed sequence is free from errors, i.e., its accuracy. A consensus distribution is derived from the assembled fragment configuration based upon the rates of sequencing errors in the individual fragments. The consensus distribution can be used to find a minimally redundant consensus sequence that meets a prespecified confidence level, either base by base or across any region of the sequence. A likelihood-based procedure for the estimation of the sequencing error rates, which utilizes an iterative EM algorithm, is described. Prior knowledge of the error rates is easily incorporated into the estimation procedure. The methods are applied to a set of assembled sequence fragments from the human G6PD locus. We close the paper with a brief discussion of the relevance and practical implications of this work.


The gene for a novel epidermal antigen maps near the neurofibromatosis 1 gene. Recently the M17S1 gene, encoding an epidermal antigen thought to play a role in cell adhesion, was mapped to chromosome bands 17q11-q12, placing it in the vicinity of the gene for the genetic disorder neurofibromatosis 1 (NF1). The pleomorphic cutaneous lesions of NF1 and the precedent for other genes being embedded within the NF1 gene prompted us to investigate whether the M17S1 gene mapped near, or within, the NF1 gene. Genetic linkage analyses revealed that M17S1 was tightly linked to NF1 and mapped within the interval bounded by D17S58 and D17S54. Physical mapping of an M17S1 cDNA on somatic cell hybrids, yeast artificial chromosomes, and an NF1 patient with a deletion involving an entire NF1 allele demonstrated that M17S1 is located at least 180 kb centromeric to the NF1 gene. The distance between the genes suggests that M17S1 is unlikely to contribute to the NF1 phenotype since a gross chromosomal rearrangement would be required to disrupt expression of both genes.


Gene order and genetic distance of 13 loci spanning murine chromosome 15. Thirteen genetic loci spanning murine chromosome 15 from 15A2 (Mlvi-2) to 15F2-3 (Gdc-1) have been mapped. The genetic distance extends to 55.4 cM. Among 151 animals, only 1 animal with a double cross-over was found. The linear order is unambiguous, with the exception of the distal end on 15F1-3. Our analysis favors the order cen-Ela-1/Hox-3-Wnt-1-Gdc-1-ter. This ordering makes necessary the introduction of three tightly spaced double recombination events around and within the Hox-3 locus. Alternatively, Hox-3 may be most distal, and several double recombinations at the telomere lead to map expansion. Despite the unequal distribution along chromosome 15 of G-versus R-bands, a comparison of distances determined by physical and genetic mapping does not indicate an overt difference in distance between both mapping techniques.


Genetic mapping of tandemly repeated telomeric DNA sequences in tomato (Lycopersicon esculentum). A telomere-associated tandemly repeated DNA sequence of tomato, TGR I, has been used to map telomeres on the tomato RFLP linkage map. Mapping was performed by monitoring the segregation of entire arrays of TGR I from a segregating F2 population using pulsed-field gel electrophoresis (PFGE). With this strategy, four telomeres have been mapped to the ends of the short arm of chromosomes 9 and 12 and the long arms of chromosomes 5 and 11, using a saturated RFLP map of tomato containing approximately 1000 RFLP markers. In all four cases, the TGR I locus maps to the end of the chromosome, and the distance between the most distal single-copy RFLP marker and the telomeric TGR I locus was between 1.6 and 9.6 cM. This indicates that the region close to the telomeres does not show an excessive rate of recombination compared to other regions of the genome and that the RFLP map of tomato is essentially complete and covers the entire genome for all practical purposes. Additionally, the mapping technique presented here should be generally applicable to the mapping of other tandemly repeated DNA sequences.


Arrangement and localization of the human GM-CSF receptor alpha chain gene CSF2RA within the X-Y pseudoautosomal region. The gene encoding one subunit of the receptor for the hemopoietic growth factor, GM-CSF, has been previously localized to the short arm of the human sex chromosomes. By pulsed-field gel electrophoresis, the precise localization of this gene, CSF2RA, within the pseudoautosomal region has been determined. The gene is located 1180 to 1300 kb from the telomere, in close proximity to the CpG island B5. The CSF2RA gene spans at least 45 kb, and a representation of most of the gene on three overlapping cosmid clones has been obtained. The exon(s) encoding the first 35 bp of cDNA sequence lies outside these cosmids. The CSF2RA gene is characterized by abundant hypervariable sequences, and a number of informative restriction fragment length polymorphisms have been defined.


Assignment of the gene for acetylcholinesterase to distal mouse chromosome 5. Characterization of genomic clones encoding mouse acetylcholinesterase enabled us to identify a restriction fragment length polymorphism that distinguishes between the progenitor strains for the recombinant inbred strain sets AKXD and BXD. The strain distribution pattern for this polymorphism indicates that Ache is located on distal mouse chromosome 5.


Genetic heterogeneity in X-linked amelogenesis imperfecta. The AMELX gene located at Xp22.1-p22.3 encodes for the enamel protein amelogenin and has been implicated as the gene responsible for the inherited dental abnormality X-linked amelogenesis imperfecta (XAI). Three families with XAI have been investigated using polymorphic DNA markers flanking the position of AMELX. Using two-point linkage analysis, linkage was established between XAI and several of these markers in two families, with a combined lod score of 6.05 for DXS16 at theta = 0.04. This supports the involvement of AMELX, located close to DXS16, in the XAI disease process (AIH1) in those families. Using multipoint linkage analysis, the combined maximum lod score for these two families was 7.30 for a location of AIH1 at 2 cM distal to DXS16. The support interval around this location extended about 8 cM proximal to DXS92, and the AIH1 location could not be precisely defined by multipoint mapping. Study of recombination events indicated that AIH1 lies in the interval between DXS143 and DXS85. There was significant evidence against linkage to this region in the third family, indicating locus heterogeneity in XAI. Further analysis with markers on the long arm of the X chromosome showed evidence of linkage to DXS144E and F9 with no recombination with either of these markers. Two-point analysis gave a peak lod score at DXS144E with a maximum lod score of 2.83 at theta = 0, with a peak lod score in multipoint linkage analysis of 2.84 at theta = 0. The support interval extended 9 cM proximal to DXS144E and 14 cM distal to F9.(ABSTRACT TRUNCATED AT 250 WORDS)


Mapping of the mouse Rxr loci encoding nuclear retinoid X receptors RXR alpha, RXR beta, and RXR gamma. Recently, a novel subgroup of nuclear hormone receptors called RXRs implicated for retinoid-mediated gene regulation have been identified. RXRs appear to interact with many other nuclear hormone receptors and modulate their functions. We have mapped genetic loci Rxra, Rxrb, and Rxrg encoding three RXR subtypes, RXR alpha, RXR beta, and RXR gamma, respectively, using interspecific backcross mice. None of the Rxr loci cosegregated with each other or with the retinoic acid receptor loci (Rar) mapped previously. Rxra mapped to Chr 2 near the centromere, Rxrb mapped to the H-2 region of Chr 17, and Rxrg was tightly linked to the Pbx gene on distal Chr 1. These results underscore that RXR genes are dispersed in the genome.


Genetic map of nine polymorphic loci comprising a single linkage group on rat chromosome 10: evidence for linkage conservation with human chromosome 17 and mouse chromosome 11. Seven genes and two anonymous markers were mapped to a single linkage group on rat chromosome 10 using progeny of an F2 intercross of Fischer (F344/N) and Lewis (LEW/N) inbred rats. Two genes, the neu oncogene or cellular homologue of the viral oncogene erbb2 (ERBB2) and growth hormone (GH) were mapped by Southern blot analysis of restriction fragment length polymorphisms. Five genes, embryonic skeletal myosin heavy chain (MYH3), androgen binding protein/sex hormone binding globulin (SHBG), asialoglycoprotein receptor (hepatic lectin)-1 (ASGR1), ATP citrate lysase (CLATP), and pancreatic polypeptide (PPY), and two anonymous markers, F16F2 and F10F1, were mapped using PCR amplification techniques. The PCR-typable polymorphic markers for the five genes were also highly polymorphic in 10 other inbred rat strains (SHR/N, WKY/N, MNR/N, MR/N, LOU/MN, BN/SsN, BUF/N, WBB1/N, WBB2/N, and ACI/N). These markers should be useful in genetic analysis of traits described in inbred rat strains, as well as in genetic monitoring of such strains. The loci in this linkage group covered 50 cM of rat chromosome 10 with the following order: MYH3, SHBG/ASGR1 (no recombinants detected), F16F2, ERBB2, CLATP, PPY, GH, and F10F1. Comparative gene mapping analysis indicated that this region of rat chromosome 10 exhibits linkage conservation with regions of human chromosome 17 and mouse chromosome 11.


Pax1, a member of the paired box-containing class of developmental control genes, is mapped to human chromosome 20p11.2 by in situ hybridization (ISH and FISH). Pax-1, a member of a murine multigene family, belongs to the paired box-containing class of developmental control genes first identified in Drosophila. The Pax-1 gene encodes a sequence-specific DNA-binding protein with transcriptional activating properties and has been found to be mutated in the autosomal recessive mutation undulated (un) on mouse chromosome 2 with vertebral anomalies along the entire rostrocaudal axis. By radioactive in situ hybridization (ISH) using a fragment from the murine Pax-1 paired box that is almost identical to the respective sequences from the cognate human gene HuP48 and fluorescence in situ hybridization (FISH) using a complete mouse Pax-1 cDNA, we have assigned the human homologue of murine Pax-1, the PAX1 locus, to chromosome 20p. The map position of PAX1 after FISH (FL-pter value of 0.34 +/- 0.04) corresponds to band p11.2. These results confirm the exceptional homology between human chromosome 20 and the distal segment of mouse chromosome 2, extending from bands F to G, and add PAX1 to the group of genes on 20p like PTPA, PRNP, SCG1, BMP2A, which are located in proximity on both chromosomes.


Mapping Creb-1 to chromosome 1 in the mouse. The gene CREB1 encoding the cyclic AMP response element DNA binding protein was previously assigned to human 2q32.3-q34. In this study, a panel of 207 backcross mice made between C57BL/10ScSn (=B10) females and (B10 x B10.L-Lsh)F1 males were used to map Creb-1 with respect to Cryg and Lsh/Vil on mouse chromosome 1. A reverse-transcribed, polymerase chain reaction-amplified cDNA probe covering bp 39 to 554 of the human sequence identified restriction fragment length polymorphisms with 7/18 restriction endonucleases used to digest whole genomic mouse DNA from the parental strains. BglII and DraI RFLPs for Creb-1 were scored on a subpanel of 16/207 known recombinants between Cryg and Lsh/Vil, yielding 2/16 recombinants between Cryg and Creb-1 and 14/16 recombinants between Creb-1 and Lsh/Vil. The 16/207 recombinants observed between Lsh/Vil and Cryg provide an estimated recombination frequency of 0.077 +/- 0.019, equivalent to a map distance of 7.7 +/- 1.9 cM. This is in good agreement with previously published map distances. The number of recombinants observed between Creb-1 and the other markers place Creb-1 approximately 1 cM distal to Cryg and 7 cM proximal to Lsh/Vil.


Chromosome 11p15 deletions in human malignant astrocytomas and primitive neuroectodermal tumors. Chromosome 11p15 deletions occur frequently in several types of human cancer, both sporadic and familial, suggesting that a tumor suppressor gene is present within the deleted chromosome region. We carried out a restriction fragment length polymorphism analysis of chromosome 11p in two types of human brain tumors: malignant astrocytoma, the most common glial tumor in adults; and primitive neuroectodermal tumor (PNET), a malignant embryonic tumor that afflicts children. Loss of heterozygosity was found in 11/43 malignant astrocytomas (26%) and in 3/11 PNETs (27%). Deletion mapping revealed a region of loss on chromosome 11p (p15.4-pter) that was common to both tumor types. To determine whether the c-H-ras gene, located on chromosome 11p in the common region of deletion, was a candidate gene, we analyzed polymerase chain reaction products corresponding to all four c-H-ras coding exons for single-strand conformation polymorphisms. The absence of electrophoretic mobility shifts in tumor DNA compared to leukocyte DNA indicated that c-H-ras gene mutations were most likely not present. These results suggested that loss of a gene on chromosome 11p15 distinct from c-H-ras is an important step in tumorigenesis within the central nervous system in both children and adults.


CD3+ T cells in severe combined immunodeficiency (scid) mice. VI. Rescue of scid-derived, IgM-producing B cells by transfer of CD4+ CD8- T cells from various lymphoid organs. Intravenous injection of purified CD4+ CD8- T cells from thymus, spleen or lymph nodes of adult dm2 donor mice (H-2d, Ld-, IgMa) into young C.B-17 scid/scid (scid) mice (H-2d, Ld+, IgMb) partially and selectively reconstituted the splenic CD4+ T-cell compartment of recipient scid mice. This was demonstrated by cytofluorographic analyses, histological examinations, growing donor-derived CD4+ T-cell lines in vitro from spleens of transplanted scid mice, and serial passage of Ld-, CD3+CD4+CD8- T-cell lines through young scid recipients for more than 1 year. Host-derived IgMb appeared in sera of all CD4+ T-cell-transplanted young scid mice, while none of the sex- and age-matched, non-transplanted control scid mice was Ig+. B-cell leakiness was most efficiently induced by transfer of low numbers of adult CD4+ CD8- thymocytes: transfer of 10(3) purified CD4+ CD8- thymocytes efficiently rescued scid-derived B cells, while transfer of 2 x 10(7) non-fractionated thymocytes from the same donor mouse was inefficient. Serum antibodies of scid mice with T-cell-induced B-cell leakiness stained congenic 'double-positive' (CD4+ CD8+) thymocytes and immunoprecipitated protein bands with an apparent MW of 14,000, 28,000, 43,000, 65,000 and 80,000 from 125I-labelled thymocytes. These data indicate that repopulation of peripheral lymphoid organs with CD4+ T cells is always accompanied by partial co-reconstitution of the B-cell system, and raises the question of the interdependence of these two lymphocyte subsets.


Subsets of null and gamma delta T-cell receptor+ T lymphocytes in the blood of young pigs identified by specific monoclonal antibodies. Rat monoclonal antibodies (mAb) against isolated pig Null T cells were derived using a novel two-colour cytofluorometric assay. One (MAC320) identified all blood CD2-sIg- 'Null' cells (present at up to approximately 6 x 10(6)/ml). Another type (MAC319 and MAC318) identified a subset comprising approximately 60% or approximately 30% of the Null cell population. This percentage appears genetically determined. This subset partially overlapped with a gamma delta T-cell receptor+ (TcR+) population which consisted of approximately 40% of Null T cells. The antibodies did not react with other leucocyte or lymphocyte populations. In non-reducing conditions, MAC320 precipitated two molecules at approximately 270,000-280,000 MW in SDS-PAGE; the larger of which was also precipitated by MAC319 (and MAC318, which binds to the same epitope). Under reducing conditions, MAC320 immunoprecipitated two or three polypeptide chains at approximately 130,000-160,000 MW; MAC319 precipitated only the largest of these polypeptides. The large MAC319+ MAC320+ molecule on one subset is removed by bromelain treatment; the smaller MAC319- MAC320+ molecule on the remaining Null cells is not bromelain sensitive. Several properties of this new antigen complex specific to pig Null T cells show that it is distinct from the ruminant T19 complex.


T cell receptor alpha-chain polymorphic allele frequencies in Caucasians and Polynesians. Restriction length polymorphisms in the variable and constant regions of the T cell receptor alpha-chain were examined in 42 Caucasians, 29 Maoris and 27 Pacific Islanders. Southern blots of Taq I digested DNA were hybridized with the T cell receptor alpha-chain probe pY14. Our results confirm that a 1.4 kb T cell receptor alpha chain-Taq 1 band is allelic to a 0.5 kb band. A significant difference in the frequency of the 1.4 and 0.5 kb alleles of the variable region of the alpha-chain was detected in Caucasians when compared with Maoris or Pacific Islanders (P < 0.0001). No differences in the frequency of the 2.0 and 7.0 kb alleles of the constant region gene were detected between any of the racial groups. These data may be relevant to ethnic differences in susceptibility to immune disorders.


Immunosuppressive factor from liver and its influence on T cell development. An endogenous immunosuppressive factor from liver, named ISF-70, of 70 kDa was studied with respect to mouse thymocyte maturation and expression of differentiation antigens in vitro. This factor induced dose-dependent alternative changes in thymocyte viability curves for 72 h incubation. Augmentation of viable cells was accompanied by a decrease in DNA synthesis and an increase in Lyt-2-positive cells. A correlation was observed between thymocyte proliferation, Lyt-2 expression and the action of ISF-70. It was found that ISF-70 distribution on thymocytes coincided with that of Lyt-2 antigen. The role of this factor in thymocyte developmental pathways and self-tolerance is discussed.


Construction of a quadroma to alpha-endorphin/horseradish peroxidase using an actinomycin D-resistant mouse myeloma cell line. A hybrid hybridoma (quadroma), secreting antibodies with double specificity to alpha-endorphin (alpha-EP) and horseradish peroxidase (HRP), has been produced. The bispecific antibodies constituted about 28-29% of all immunologically active IgG, produced by quadroma. The quadroma was isolated by fusion of two mouse hybridomas (anti-HRP and anti-alpha-EP) with distinct phenotypes: double mutant AMDR/HAT(S), and wild type (AMDS/HATR). A novel strategy for the construction of a double-mutant was applied, based on the use of an actinomycin D-resistant (AMDR) mouse myeloma for initiation of one of the parental hybridomas.


Thy-1 antigen-mediated adhesion of mouse lymphoid cells to stromal cells of haemopoietic origin. Thy-1 antigen is one of the widespread cell surface antigens. The antigen is expressed in the cells of a wide variety of different tissues and species and its pattern varies considerably during development and among species. Although the Thy-1 is one of the members of Ig-superfamily its function(s) remain unknown. Here we show that monoclonal antibodies to mouse Thy-1.2 antigen prevent adhesion of Thy-1.2-positive mouse T-leukemic cells 127 to monolayers of two mouse bone marrow stromal cell lines (14F1.1 and MBA-15). The same antibodies do not prevent adhesion of cell 127 to monolayers of two other mouse bone marrow stromal cell lines (MBA-2.1 and MBA-1.1.1) and to a wide spectrum of different normal and tumor mouse cell lines. A characteristic of the cell line 14F1.1 is the ability to support growth of mouse haemopoietic stem-cells. This data suggest that cells of stromal lines 14F1.1 and MBA-15 contain on their surface a specific ligand for the Thy-1 molecule. Cell-cell interaction via this ligand and the molecule on the cell surface may be an important step for development of certain lineages of cells.


Apparently non-expressed alleles of factor B (BF) code for hypomorphic proteins. In three families with an apparent non-expressed factor B (BF) allele (BF*Q0), advanced methods of isoelectric focusing for the determination of BF F subtypes revealed different hypomorphic BF products (BF QL) with functional hemolytic activity expressed by the assumed BF*Q0 allele. A Taq I and a Msp I restriction fragment length polymorphism as well as the Ba fragment of the expression products showed banding patterns for the BF*QL alleles corresponding to BF S types, whereas an altered Bb fragment was seen in two BF QL products. In one family an intragenic recombination site within the Bb part of the BF gene was assumed. Investigations of factor B and its conversion fragments, as demonstrated by the used methods, allow to complement molecular genetic investigations of BF*Q0 alleles in heterozygous genotypes on a protein level. We conclude that apparently non-expressed alleles of factor B code for hypomorphic but functionally active proteins.


Antihistaminic efficacy of Ranitidine with & without Dimethendine maleate on histamine-induced cutaneous reactions. The effect of Ranitidine, the H2-receptor antagonist, was investigated on cutaneous response to intradermal injection of histamine in healthy volunteers in a controlled, randomized, cross over study. The response was compared with that of the H1-receptor blocker; Dimethendine maleate used alone and in combination with the two antagonists. Reduction in the wheal area was significant in subjects pretreated with Ranitidine alone (P less than 0.05), and Dimethendine maleate alone (P less than 0.05); the combination of the two antagonists, did not produce additional reduction. Reduction in erythema area was not significant with Dimethendine maleate alone, but significant with Ranitidine alone (P less than 0.01). With the combination of the two antagonists the reduction was not more significant than with Ranitidine alone. The flare response scoring on visual analogue scale was not reduced significantly by Dimethendine maleate alone but reduced significantly by Ranitidine alone (P less than 0.10), and by combination of Ranitidine and Dimethendine maleate (P less than 0.05). Thus, Ranitidine appears to be more effective than Dimethendine maleate in reducing the erythema area and intensity of flare response and equieffective in reducing wheal response to histamine injection.


Whole body autoregulation in reduced renal mass hypertension. Whole body autoregulation in conscious rats can be shown in the absence of the rapid acting neural and hormonal controllers of blood pressure. It is hypothesized that this phenomenon is responsible for the gradual rise of vascular resistance observed in volume-dependent forms of hypertension such as reduced renal mass-salt-induced hypertension. To examine the hypothesis, we evaluated the gain of whole body autoregulation at various stages of reduced renal mass hypertension to determine if acute autoregulatory capacity is altered during chronic hypertension. Rats underwent reduced renal mass surgery (nephrectomy plus 70% reduction of remaining kidney) and were studied at 2 (n = 8), 4 (n = 6), and 6 (n = 7) weeks after high salt diet. Control rats (n = 6) underwent nephrectomy and sham surgery and were studied after 2 weeks of high salt diet. All reduced renal mass rats showed progressive hypertension (2 weeks, 136 +/- 5; 4 weeks, 157 +/- 8; and 6 weeks, 171 +/- 10 mm Hg) compared with sham rats (113 +/- 4 mm Hg). We observed an increase in basal level of total peripheral resistance index after neurohumoral blockade in reduced renal mass rats (2 weeks, 1.64 +/- 0.06; 4 weeks, 1.79 +/- 0.10; and 6 weeks, 1.89 +/- 0.09 mm Hg.100 g-1.min-1.ml-1) compared with sham rats (1.56 +/- 0.10 mm Hg.100 g-1.min-1.ml-1).(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelial role in ouabain-induced contractions in guinea pig carotid arteries. The influence of endothelium on the direct contractile effects of ouabain in vascular smooth muscle was analyzed in isolated perfused guinea pig carotid arteries. After blocking the neurogenic component of the glycoside contraction with alpha-adrenergic receptor blocking drugs or treating the animals with reserpine, ouabain-induced contractions were markedly reduced in vessels with intact endothelium. However, removal of the vascular endothelium from reserpinized carotid arteries resulted in ouabain-induced contractions similar to those observed in control arteries. These effects were not mimicked by the inhibitor of nitric oxide NG-monomethyl L-arginine or by the cyclooxygenase blocker indomethacin. Bioassay experiments suggested that these endothelial effects are mediated by diffusible factors. Uptake of 86Rb to measure sodium pump activity was significantly reduced by removal of the endothelium. These results suggest the existence of an inhibitory modulation by the endothelium of contractions induced by ouabain, likely mediated by a diffusible factor (or factors) released from these cells. The nature of this substance is unknown, but it is neither related to prostaglandins nor a nitric oxide-related compound. Its mechanism of action could be the stimulation of vascular sodium pump activity, the antagonism of the pump's inhibition by ouabain, or both.


Immunohistochemical demonstration of androgen receptors on testicular blood vessels. Using an antiserum directed against the human and rat androgen receptor we have used immunohistochemistry to demonstrate that nuclear androgen receptors are present in the muscular layer of almost all arteries within the rat testis. These receptors are apparently influenced by testosterone. They disappear after Leydig cell depletion induced by ethanedimethane sulphonate (EDS) and return after testosterone treatment. It is suggested that testicular blood vessels could be a target-organ for androgens and may mediate some of the effects of androgens on the testicular microcirculation.


Suppression of basal, PMA- and IFN-alpha-, but not IFN-gamma-induced expression of HLA class I in v-myc-transformed U-937 monoblasts. Recent studies have suggested that certain oncogenes, in particular members of the myc family, may be involved in the down-regulation of HLA class-I antigen expression observed in many types of tumor. We report that constitutive expression of an OK10 v-myc gene in human monoblastic U-937 cells results in a reduced expression of HLA class-I cell-surface expression and decreased levels of HLA class-I protein and mRNA. All class-I alleles, with the possible exception of HLA A3, were affected, as shown by one-dimensional isoelectric focusing (ID-IEF). Basal expression of the beta 2m chain was also reduced, although to a lesser extent. In addition, we show that the PMA-, and at least partially the IFN-alpha-induced increase in HLA class-I antigen expression, was inhibited in U-937-myc cells both at the protein and the mRNA level. In contrast, the response to IFN-gamma was normal. Another important difference in the response to IFN-gamma and alpha was that, while IFN-gamma abrogated the v-myc block of PMA-induced differentiation of U-937 cells, as previously reported, IFN-alpha did not. Our data show that v-myc negatively affects the regulation of both basal and inducible HLA class-I antigen expression.


Treatment of liver metastases of human colon cancers in nude mice with somatostatin analogue RC-160. Hepatic metastases of colon 320 DM and WidR human colon cancers in nude mice were treated by s.c. injections of somatostatin analogue RC-160 for 4 weeks. Chronic administration of RC-160 significantly inhibited the incidence and growth of liver metastases of these 2 colon-cancer cell lines. After RC-160 treatment, the incidence of liver metastases decreased by 25% for colon 320 DM cells and by 37.5% for WidR cells. The mean number of metastatic tumors in each liver decreased by 47.9% for colon 320 DM and 42.6% for WidR. Survival times of mice with liver tumors of colon 320 DM and WidR cells were prolonged by 20 days and 7 days, respectively. The inhibitory effect of RC-160 on the growth of these 2 colon cancers implanted s.c. was also observed. After administration of RC-160 for 4 weeks, the mean tumor volume in the treated groups was only 39.8% of that of controls for the colon 320 DM line and 58% for the WidR line. Tumor-growth rate and final tumor weight were also significantly decreased, while tumor-volume doubling time and tumor-growth delay time were prolonged. The effect of RC-160 on cellular proliferation in the tumors was studied by in vivo labelling with bromodeoxyuridine and immunoperoxidase staining. The mean labelling index in the treatment group was reduced by 14.9% and 19.5%, respectively, for colon 320 DM and WidR tumors. The cytostatic effect of RC-160 was also evident from the apparent reduction in DNA and protein content in the tumor tissues of these cancer lines. Our findings suggest that somatostatin analogue RC-160 may be useful for the treatment of patients with hepatic metastases of colon cancer.


Atrial fibrillation and flutter after coronary artery bypass surgery: epidemiology, risk factors and preventive trials. Atrial fibrillation and atrial flutter are common arrhythmias after coronary artery bypass grafting. Although the consequences of the arrhythmia are generally not life-threatening, it constitutes a major clinical problem often requiring conversion to sinus rhythm. Atrial fibrillation or flutter can result in hypotension, heart failure, pneumonia, and stroke. This article reviews the literature on epidemiology, electrophysiology, risk factors, and preventive trials. The major conclusions are: (1) In patients undergoing coronary artery bypass surgery, the incidence of postoperative atrial fibrillation or flutter is 20-30%, the peak incidence being on the second or third postoperative day. (2) The strongest independent preoperative predictor for atrial fibrillation or flutter is the patients' age. (3) Intra-atrial conduction delay recorded pre and peroperatively may predict development of atrial fibrillation. (4) Peroperative inducibility of atrial fibrillation by pacing the right atrium may identify patients at risk for postoperative atrial fibrillation. (5) Development of postoperative atrial fibrillation or flutter has not been associated with peroperative or postoperative events. (6) The specificity and sensitivity of age and other possible relevant factors for prediction of atrial fibrillation or flutter after coronary artery bypass grafting is low. (7) No effective prophylactic regimen has yet been established.


Pulmonary involvement in nephropathia epidemica as demonstrated by computed tomography. In a prospective study 19 adult patients with nephropathia epidemica were examined in the acute phase of disease with computed tomography (CT) of the lungs and conventional chest radiography. Infiltrates and/or pleural effusions were seen in ten of 19 patients. In two of the patients, abnormalities were disclosed only by CT. Patients with pathologic radiography findings had a more pronounced inflammatory response, as measured by C-reactive protein and leukocyte count, than did those with normal radiography findings. It is concluded that radiological evidence of pulmonary involvement is a common finding early in the course of nephropathia epidemica. The possibility that the lung may be a site of viral replication merits further investigation.


S-fimbriae mediated adhesion of Escherichia coli to human buccal epithelial cells is age independent. S-fimbriated Escherichia coli, which cause sepsis and meningitis in the newborn, bind to sialic acid-containing glycoprotein structures on the surface of human buccal epithelial cells. The dependence of this binding on host age was examined. S-fimbriated E. coli adhered in comparable numbers to cells in newborns, infants, children and adults (23.0 +/- 8.6; 23.1 +/- 11.5; 24.7 +/- 7.9; 28.9 +/- 8.8). Thus, the increased susceptibility of neonates to infections caused by S-fimbriated E. coli cannot be explained by enhanced adhesion to epithelial cells.


Amlodipine in patients with stable angina pectoris treated with nitrates and beta-blockers. The influence on exercise tolerance, systolic and diastolic functions of the left ventricle. The effects of 5 and 10 mg of amlodipine and of placebo were compared in 21 patients with stable angina pectoris and multivessel coronary artery disease. The blind comparison was performed by means of bicycle ergometry and stress echocardiography using esophageal stimulation of the left heart atrium. All patients subsequently received placebo, amlodipine 5 mg and 10 mg for 2 weeks. In bicycle ergometry both doses of amlodipine in comparison with placebo significantly lowered the ST segment depression in lead V5 and prolonged the time to onset of angina. The exercise duration was significantly prolonged only after 10 mg of amlodipine. In stress echocardiography 10 mg of amlodipine significantly improved ejection fraction and reduced wall motion score during stimulation and increased peak velocity of relaxation of left ventricular posterior wall at rest and immediately after stimulation. In the patients with left ventricular end-diastolic pressure < or = 20 mmHg, amlodipine reduced the ratio of peak transmitral flow velocity in atrial contraction to that in early diastole (A/E) at rest and shortened deceleration time at rest and immediately after stimulation. Amlodipine in patients with stable angina pectoris significantly improved the exercise tolerance and the function of the left ventricle in a dose-dependent way. Amlodipine was well tolerated.


Combined two-dimensional and Doppler echographic examination of internal mammary artery grafts from the supraclavicular fossa. The noninvasive examination of internal mammary artery grafts is gaining importance with the increasing use of this vessel in the surgical treatment of coronary atherosclerosis of the left anterior descending artery. We studied 36 patients (37 internal mammary artery grafts) with combined two-dimensional and pulsed Doppler echography from the supraclavicular fossa. Adequate visualization and Doppler signals were obtained in 95% of arterial grafts. Twenty-four grafts leading to an area without evidence of old myocardial infarction or ischemia and 10 grafts leading to an area of old myocardial infarction but without evidence of ischemia on exercise showed a significant decrease of the peak systolic velocity and of the peak systolic velocity/peak diastolic velocity ratio as compared to the controls, which consisted of the contralateral internal mammary arteries in situ. One patient with a distally subtotally occluded mammary artery graft had a flow pattern different from the other bypassed mammary arteries. It seems that combined two-dimensional and pulsed Doppler echography is a useful method to evaluate the functional status of internal mammary artery grafts.


Open trial of tacrine therapy in 70 HIV-infected patients. The purpose of this study consisted in following-up the biological and clinical parameters in HIV infected patients treated with tacrine (THA). THA (150-300 mg/d) was administrated to 70 patients (39 IVC I and 31 IVC II and III). Thirty-five were treated after discontinuation of AZT treatment and 35 as a first intention treatment. Thirty (43%) patients showed an increase in the CD4+ cell count by more than 50% relative to pretreatment levels and fifteen (21%) showed an increase of more than 25%. p24 antigenemia (Ag p24) became negative in eight of the twenty-seven patients who were initially positive, and decreased by 25 and 50% in nine and six patients, respectively. Ag p24 was therefore decreased in 80% of the patients. From a clinical point of view, there were two deaths (3%) and five opportunistic infections (7%). The treatment with THA was stopped in five patients because of side effects (nausea, rash). Neither hepatotoxicity, hematotoxicity, nor pancreatitis was observed during the THA treatment. In group II and III only two patients (6%) developed an opportunistic infection.


Pharmacokinetic/pharmacodynamic characteristics of the beta-2-agonists terbutaline, salbutamol and fenoterol. The clinical pharmacokinetics and pharmacokinetic/dynamic properties of the beta-adrenergic drugs fenoterol, salbutamol and terbutaline are reviewed. Sulfate conjugates are the main metabolites in man. The protein binding of these derivatives is rather weak with most pronounced binding observed e.g. fenoterol (40%). Disposition after parenteral administration shows a multi-exponential behavior for all the substances with linear but also stereo-selective pharmacokinetics. After parenteral administration, the drugs are mainly eliminated by renal processes while after oral administration a pronounced metabolic clearance (high first pass effect) is responsible for a low bioavailability, especially for fenoterol (2%). The total clearance for fenoterol is about twice that of salbutamol and terbutaline. Seven to 15% of the delivered aerosol reach typically the systemic circulation. In patients with respiratory disorders, pulmonary absorption is however highly dependent on the disease state. Pharmacokinetics in children do not significantly differ from adults when expressed per kg body weight. Patients with renal failure but not asthmatics show changed pharmacokinetic profiles. Only insignificant interactions with other drugs have been found. Pharmacokinetic/dynamic modeling approaches indicated that fenoterol is 25 times more active at the site of action than salbutamol and terbutaline, but all three drugs show similar bronchopulmonary selectivities. When the overall clinical activity, determined by pharmacokinetic and dynamic properties is compared, the activity gap is reduced: fenoterol (8) greater than salbutamol (2) greater than terbutaline (1). Differences in the first pass effect even inverse the pattern after oral administration. PK/PD modeling quantified the pulmonary effect after inhalation and suggested that the higher incidence of side effects for fenoterol might be linked to an overdosing problem. The application of PK/PD principles may improve the clinical usage and therapy of beta-2-adrenergic drugs.


Laparoscopy bashing.

Laparoscopy bashing. Therapeutic laparoscopy--operative laparoscopy--has been employed in increasing proportion to laparotomy for various gynecological pathologies. Although routinely used for sterilization procedures, acceptance first for tubal pregnancy and then for other adnexal surgery has been gained. Now advocates are using the laparoscope for assisted hysterectomy, lymph node dissections, and other questionable indications. Coupled with the emergence of rapidly changing technology is the problem of adequate teaching of postgraduate physicians and proper credentialing of individuals, especially in smaller community hospitals.


Ovarian consequences of the transient interruption of combined oral contraceptives. The combined oral contraceptive pill is an efficient means of contraception. It acts at different levels of the genital tract. Despite its efficiency, it is universally suggested that patients take the pill at regular daily intervals. Little attention has been given to the question of what happens if you miss the pill one day or more. A study was undertaken to evaluate the consequences of pill misses at different times of the cycle. Forty-seven young, healthy, normally menstruating patients voluntarily enrolled. All were given Cilest (ethinyl estradiol 35 micrograms and norgestimate 250 mg, Cilag France) for 21 days without any misses. Then, after a 7-day interval, they were prescribed one (group 1), two (group 2), three (group 3) or four days of pill misses, to occur respectively on day 1 (group a), 6 (group b), 12 (group c) or 18 (group d) of a new 21 day cycle; supplementary contraceptive means were recommended. Four patients had no miss prescribed and served as controls. Ovarian function was evaluated with daily estrogen measurements (E1 + E2 enzymatic dosage, BioMérieux, France) and ultrasound examinations. When required, because of significant increase in estrogen or because of follicular growth detected on ultrasound, LH and progesterone were measured. None of the patients experienced a normal ovulation. Four patients (1 control, 1 from group 2a, and 2 from group 3a) had a significant increase in estrogen levels and had a follicular image on ultrasounds. One of them (group 3a) had a follicular rupture, but none had a LH surge or increase in progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)


Combined therapy with bromocriptine and clomiphene citrate for patients with normoprolactinemic amenorrhea. The purpose of this study was to investigate the combined effect of bromocriptine (Brc) and clomiphene citrate (Cl) treatment on 40 patients with normoprolactinemic amenorrhea who failed to respond to Cl alone. The ovulation rate in this treatment was 57.3% (23/40) in the 40 cases, 55.6% (99/178) in 178 cycles; the pregnancy rate was 26.7%. This treatment was effective in 14 of 21 women with polycystic ovary-like syndrome (66.7%). Among those women who responded to treatment, prolactin (PRL) and LH levels were significantly decreased. Estradiol and progesterone levels were significantly increased in the patients who responded. Before treatment, the responsiveness of LH to LHRH among responders to Brc/Cl therapy was significantly higher than among the nonresponders. After treatment, the LH-releasing response following a conjugated estrogen injection in the patients who responded to the treatment was significantly greater than that in the patients who did not respond. The results suggest that the therapeutic effect of this treatment may be primarily due to the restoration and improvement of the impaired hypothalamo-pituitary axis.


Müllerian remnants in complete androgen insensitivity syndrome. Patients with complete androgen insensitivity syndrome rarely have Müllerian remnants. A patient with this syndrome found to have a microscopic fallopian tube at the time of gonadectomy is described and possible etiologies for the finding are discussed.


Precursors of molecules related to mammalian opioid peptides in brain of a marine worm. Total mRNA were extracted from brain of Nereis diversicolor (Annelida, Polychaeta) and were translated in vitro or in ovo. The newly synthesized polypeptides were analyzed through electrophoresis of immunoprecipitated products or the Western blotting technique using polyclonal antibodies raised against mammalian dynorphin 1-17 and mammalian alpha-neo-endorphin. Among the products translated in vitro, only one class of polypeptide of 70 kDa was recognized by anti-dynorphin 1-17 antibodies. Furthermore, some in ovo translated products as well as proteins extracted from brain of worms showed identical immunoreactivity. These polypeptides, 60-70 kDa, reacted with anti-dynorphin 1-17 and anti alpha-neo-endorphin antibodies. These results suggest the existence of epitopes common to in ovo and in vitro translated products, to polypeptides extracted from the brain and to some mammalian opioid peptides of the prodynorphin family. We postulate the presence, in the brain of N. diversicolor, of precursors of peptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Data reported in this investigation do not allow us to propose or even postulate the presence, in the brain of the worm, of one precursor molecule common to polypeptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Furthermore, the Nereis precursor molecules exhibit a clear-cut difference in molecular mass with the mammalian prodynorphin: 70 kDa versus 30 kDa.


Prevalence of polycystic ovaries by transvaginal ultrasound and serum androgens. The present study compares the ovarian area and the androgen levels (testosterone, free testosterone, SHBG, testosterone/SHBG ratio) in the two patterns (peripheral cystic pattern, general cystic pattern) of polycystic ovaries diagnosed by transvaginal ultrasound. The results were as follows. (1) The ovarian area of the peripheral cystic pattern group of polycystic ovaries was significantly greater than that of normal controls, but in the general cystic group it was not. (2) Serum levels of testosterone and free testosterone, and the testosterone/SHBG ratio in patients with polycystic ovary syndrome were significantly higher than those of healthy controls; but with ultrasonographic classification, there was no significant difference between the peripheral cystic and general cystic groups. (3) In the polycystic ovary syndrome, elevated levels of testosterone and free testosterone, and of the testosterone/SHBG ratio were observed in approximately half of the patients. Ovarian morphology was different, but androgen status was similar in the two patterns of polycystic ovaries.


Reduced peptide bond cyclic somatostatin based opioid octapeptides. Synthesis, conformational properties and pharmacological characterization. The conformational and pharmacological properties that result from peptide bond reduction as well as the use of secondary amino acids in a series of cyclic peptides related to the mu opioid receptor selective antagonist D-Phe1-Cys2-Tyr3-D-Trp4-Orn5-Thr6-Pen7+ ++-Thr8-NH2 (IV), have been investigated. Peptide analogues that contain [CH2NH] and [CH2N] pseudo-peptide bonds (in primary and secondary amino acids, respectively) were synthesized on a solid support. Substitution of Tyr3 in IV by the cyclic, secondary amino acid 1,2,3,4-tetrahydroisoquinoline carboxylate (Tic) and of D-Trp4 with D-1,2,3,4-tetrahydro-beta-carboline(D-Tca4), gave peptides 4 and 1, respectively. Both analogues displayed reduced affinities for mu opioid receptors. Conformational analysis based on extensive NMR investigations demonstrated that the backbone conformations of 1 and 4 are similar to those of the potent and selective analogue D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (I), while the conformational properties of the side chains of Tic3 (4) and D-Tca4 (1) resulted in topographical properties that were not well recognized by the mu opioid receptor. Peptide bond modifications were made including (Tyr3-psi[CH2NH]-D-Trp4), 3; (Tyr3-psi[CH2N]-D-Tca4), 2; and (Cys2-psi[CH2N]-Tic3), 6. These analogues showed decreases in their mu opioid receptor affinities relative to the parent compounds IV, 1, and 4, respectively. 1H NMR based conformational analysis in conjunction with receptor binding data led to the conclusion that the reduced peptide bonds in 2, 3, 5, and 6 do not contribute to the process of discrimination between mu and delta opioid receptors, and in spite of their different dynamic behaviors (relative to 1 and 4), they are still capable of attaining similar receptor bound conformations, possibly due to their increased flexibility.


Endoscopic carbon dioxide laser ovarian wedge resection in resistant polycystic ovarian disease. Clomiphene citrate therapy has been found to improve the infertility rate in women suffering from polycystic ovarian disease (PCOD). However, there still exists a group of women with PCOD who fail to respond to clomiphene citrate or human menopausal gonadotropin/human chorionic gonadotropin or urofollitropin/chorionic gonadotropin treatment, an ovarian wedge resection by laparotomy approach which has been known for 50 years. Within the last decade, translaparoscopic electrocautery and laser drilling techniques have been utilized. In 1986, a translaparoscopic carbon dioxide laser ovarian wedge resection was introduced for resistant PCOD. This paper describes the surgical principle of a translaparoscopic carbon dioxide laser ovarian wedge resection. This type of treatment results in a 75% crude conception rate, with a 67% rate of healthy live birth. There was an 8% postsurgical adhesion rate among 12 cases that were incorporated into the study. This mode of therapy may prove to be very useful, safe, easy to perform, and cost effective as a second-line therapy for resistant PCOD in cases where medical inductions fail to achieve ovulation.


Interaction of glucagon with artificial lipid bilayer membranes. The enhancement of fluorescence emission from the tryptophan residue of glucagon, the quenching of that emission with acrylamide and with 5-doxyl and 16-doxyl stearic acid, circular dichroism spectra, the release of 6-carboxyfluorescein, and polarized infrared attenuated total reflection (IR-ATR) spectra were used to study the interaction of glucagon with intact lipid vesicles and flat bilayers. Dimyristoylphosphatidylcholine bound the peptide only below the main transition temperature, thus confirming earlier results of Epand et al. (1977). However, the peptide is also bound by vesicles of unsaturated lipids above their transition temperature, suggesting an influence of lipid area on the binding process. Circular dichroism showed that binding to such vesicles also increases the helix content of glucagon. The IR-ATR study and a comparison with dynorphin-A-(1-13)-tridecapeptide revealed profound differences in orientation of the two peptides. The dichroic ratios and the derived order parameters indicated an isotropic orientation of the helical segments of glucagon, but did not exclude a principal orientation of the molecules lying flat on the membrane surface. In contrast, the axis of the dynorphin helix is clearly oriented normal to the interface. The two peptides also differ in their rates of 6-carboxyfluorescein release, suggesting a deeper penetration of the primary amphiphilic helix of dynorphin A-(1-13) than of the secondary amphiphilic helix of glucagon.


Etiological factors of male infertility in Africa. Of the 595 infertile African males studied, 192 (30.8%) were azoospermic and 413 (69.40%) had oligospermia. Azoospermia was caused by obstruction to the vas and/or epididymis in 44% of cases and testicular lesions in the remaining 56% of cases, whilst the oligospermia was probably caused by obstruction in 4.7% of cases and testicular lesions in 85.3%. Bilateral testicular biopsies were performed on 302 patients. A variety of pathological conditions were observed; the most prevalent was hypospermatogenesis, in 12% of cases. A significant portion (37.2%) of patients without testicular biopsies had clinically detectable testicular or epididymal abnormalities. There was a higher incidence (12%) of inflammatory testicular or prostatic conditions in this study as compared with those found in Europeans, suggesting that inflammatory conditions contribute more to male infertility in Africa. Only a single case of chromosomal abnormality was detected.


N-alkoxycarbonyl-glutamic and aspartic acids. Studies on the activation and cyclodehydration and side-reaction encountered in analysis of glutamic acid using Fmoc-chloride. N-Alkoxycarbonylaminodicarboxylic acids were reacted in dichloromethane with N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride, and with methyl chloroformate in the presence of N-methylmorpholine. Removal of secondary products by washing the mixtures with aqueous solutions gave good yields of the pure crystalline internal anhydrides. Anhydrides of N-benzyloxycarbonyl- (Z) and N-9-fluorenylmethoxycarbonyl-(Fmoc) L-glutamic and L-aspartic acids and of N-tert.-butoxycarbonyl-L-aspartic acid were prepared in this way. The compounds were shown to be amenable to normal phase high-performance liquid chromatography (NP-HPLC) on a CN-column using tert.-butanol-hexane as solvent. The products of the reactions of Z- and Fmoc-glutamic acid with hot acetic anhydride were examined by nuclear magnetic resonance and NP-HPLC before and after methanolysis in an attempt to establish if any of the corresponding pyroglutamates were formed. The reaction of Fmoc-chloride with Fmoc-glutamate was examined for the same reason. It is concluded that the side product generated during the reaction of Fmoc-chloride with glutamic acid which is used for analysis of the latter is the N-protected internal anhydride and not the pyroglutamate as reported in the literature.


Conformational studies of N-Tyr-MIF-1 in aqueous solution by 1H nuclear magnetic resonance spectroscopy. N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) is an endogenous brain peptide with multiple effects on animal behavior. However, there have been no studies on the conformation of this tetrapeptide. In this report, we studied the conformation of N-Tyr-MIF-1 in aqueous solution by conventional one-dimensional and two-dimensional (COSY and NOESY) 1H nuclear magnetic resonance spectroscopy at 300 MHz. A complete set of assignments for the resolved resonances and approximate assignments for the overlapping resonances were made. The results demonstrate that N-Tyr-MIF-1 is in slow exchange between two conformers, most likely determined by the cis and trans states of the proline residue. The minor conformation represents 30 +/- 3% of the population over the temperature range from 3 degrees to 73 degrees. In the major conformation, the tyrosine aromatic ring appears to be close enough to interact directly with the proline pyrrolidine ring, as indicated by a strong temperature dependence of the proline C beta H, C delta H and C delta H' chemical shifts. In contrast, this interaction of the tyrosine and proline rings is not present in the minor conformation.


Flow cytometric comparison between swim-up and Percoll gradient techniques for the separation of frozen-thawed human spermatozoa. Viable human spermatozoa were recovered from cryopreservation by either swim-up or Percoll density gradient techniques and their cell-surface oligosaccharide components were compared to those of fresh sperm. Sperm were labeled with FITC-Con A and analyzed by fluorescence microscopy and flow cytometry. By fluorescence microscopy, fresh sperm were uniformly labeled in the neck region alone. Frozen-thawed sperm, obtained by either swim-up or Percoll density gradients, showed two patterns of staining: one sperm population was stained in the neck region only, whilst another showed staining in both the neck and acrosome regions. No difference between sperm recovered by the two techniques was apparent. Flow cytometry profiles of fresh sperm revealed a single peak of fluorescence, whereas cryopreserved sperm gave two peaks of fluorescence. Samples recovered by Percoll density gradient contained significantly more sperm with the same fluorescence pattern as that observed for fresh sperm than did those recovered by swim-up. Cryopreservation alters the cell-surface oligosaccharide components of spermatozoa. Recovery of cryopreserved sperm by Percoll density gradient yields a greater proportion of sperm which resemble fresh sperm than does swim-up.


Plasminogen activator: the identification of an additional proteinase at the outer acrosomal membrane of human and boar spermatozoa. We have recently shown that spermatozoa of various species contain both types of plasminogen activator, the tissue-type (t-PA) and the urokinase-type (u-PA). In the present study, the localization of t-PA and u-PA in plasma membrane and outer acrosomal membrane of human and boar spermatozoa has been investigated. The identification of the type of the plasminogen activator (t-PA or u-PA) was made immunologically. In human spermatozoa, the outer acrosomal membrane and plasma membrane contained both types of plasminogen activator (t-PA and u-PA); in addition, t-PA antigen was measured. In boar spermatozoa, the outer acrosomal membrane contained only t-PA, whereas plasma membrane contained both types of plasminogen activator (t-PA and u-PA). Plasminogen activator inhibition (PAI) has also been demonstrated in plasma and outer acrosomal membranes of both species and identified as PAI-1 in membranes of human spermatozoa.


Apraclonidine and clonidine: a comparison of efficacy and side effects in normal and ocular hypertensive volunteers. We performed a prospective, randomized double blind study comparing the cardiovascular and intraocular pressure (IOP) effects of unilateral therapy with clonidine 0.125% and apraclonidine hydrochloride 1.0% in 15 normal and 15 ocular hypertensive volunteers. Baseline values were obtained prior to instillation. One drop of test medication (clonidine, apraclonidine or placebo) was instilled unilaterally, and the post-instillation measurements were taken at 1, 2, 4, 6 and 8 hours. Apraclonidine 1% produced a maximum 31.4% +/- 6.9% (4.83 +/- 1.17 mmHg) decrease in mean IOP in ocular normotensive volunteers and 33.9% +/- 6.9% (10.10 +/- 2.45 mmHg) in ocular hypertensive patients (p < 0.001). These values were 22.1% +/- 6.9% (2.90 +/- 1.94 mmHg) and 22.7% +/- 6.9 (6.80 +/- 2.31 mmHg), respectively in clonidine group (p < 0.001). In apraclonidine group, there were no changes in contralateral IOP, blood pressure or pulse rate. Clonidine produced a significant decrease in contralateral IOP, but this reduction was not statistically significantly different than that of placebo. In clonidine group, there was no change in pulse rate, but a significant decrease in blood pressure. Eyelid retraction, conjunctival blanching and mydriasis were noted in eyes treated with apraclonidine. However there were no statistically and clinically significant changes in pupil size or interpalpebral fissure width with clonidine. This study suggests that apraclonidine appears to be safer and more effective ocular hypotensive agent than clonidine in treatment of glaucoma.


Dopamine, dopaminergic drugs and ocular hypertension. The study refers to the clinical experiences performed with several D1 and D2 dopaminergic receptors agonists in 20 patients with high tension open angle glaucoma. The substances were administered topically as eye drops as well as an ocular eye bath. The parameter examined was intraocular pressure (IOP). The substances taken in consideration were: Dopamine, Ibopamine (dopamine analog), Fenoldopam and 3B90 (D1-receptor agonists) and Bromocriptine (dopaminergic agonist with higher affinity for D2 than for D1-receptors). It has been shown that all selective D1-receptors agonists induce a significant increase in IOP only in eyes with hydrodynamic disorders (p < 0.001). Such hypertensive effects could not be antagonized either by topically administered dopaminergic antagonists (Sulpiride, D2-receptors antagonist, and Haloperidol, non-selective dopaminergic antagonist) or by the pretreatment with the commonly used topical antiglaucomatous drugs. The only substance which proved able to inhibit the IOP increase induced by the D1-receptors agonists was the D1-selective antagonist SCH-23390, suggesting that IOP increase may be a result of a stimulation of the D1-receptors. The authors hypothesize that dopaminergic system may play a role in the regulation of aqueous humor hydrodynamics.


Human leukocyte antigen serologic and DNA typing of Behçet's disease and its primary association with B51. Ninety Japanese patients with Behçet's disease (BD) were typed for human leukocyte antigen (HLA)-DRB1, -DQA1-, -DQB1, and -DPB1 alleles by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and for HLA-A, -B, -C, -DR, and -DQ antigens by conventional serologic typing. Serologic HLA typing showed a remarkably significant increase of HLA-B51 and a significant decrease of HLA-DQw1 in the patients with BD, especially those with ocular lesions including complete type, as compared with the control group (for B51, chi-squared = 46.75, P corrected < 0.001, relative risk [RR] = 7.9; for DQw1, chi-squared = 12.10, P corrected < 0.01, RR = 0.4). By PCR-RFLP genotyping, no significant difference was revealed in any class II alleles between the patient and the control groups in the corrected P value test, but P value analysis showed the significantly high frequency of DRB1*0802 and the significantly low frequencies of DQA1*0103, DQB1*0601, and DQB1*0501. No significant difference was observed in any DPB1 alleles by either P value analysis. These results indicated that the primary and primordial gene(s) responsible for the susceptibility to BD, especially related to ocular lesions, were not located in the HLA class II gene region but were in or very close to the HLA-B locus in the class I region. They also suggested the possibility that BD was a symptom complex associated with some independent diseases.


MK-801 protects retinal neurons from hypoxia and the toxicity of glutamate and aspartate. The protective effect of the anticonvulsant MK-801 and the antitussive dextromethorphan, which are both N-methyl-D-aspartate receptor antagonists, and kynurenic acid, a broad-spectrum excitotoxin antagonist, was tested in cultured rat retinal cells in an hypoxic environment. The protective effect of these antagonists also was tested in cultured retinal cells and in intact adult rat retinas exposed to the exogenous excitotoxins L-glutamic acid and N-methyl-D-aspartic acid. MK-801 and kynurenic acid protected retinal neurons from hypoxic damage and from the toxicity of exogenous L-glutamic acid and N-methyl-D-aspartic acid. Dextromethorphan, a less potent antagonist, did not protect the retinal neurons from hypoxic damage or the toxicity of exogenous L-glutamic acid, but did attenuate N-methyl-D-aspartate toxicity. These results provide evidence that the synaptic release of excitatory transmitters, most likely glutamate and aspartate, mediate the death of hypoxic retinal neurons. Compounds related to MK-801 may have possible therapeutic applications in the management of retinal ischemia.


[Elastosis perforans serpiginosa. Considerations on the pathogenesis based on a typical case]. Elastosis perforans serpiginosa (EPS) is a rare entity belonging to the group of primary perforating dermatoses. A 13-year-old male patient with Down's syndrome developed reddish hyperkeratotic papules in a serpiginous and ellipsoid configuration on the face. Histological examination revealed transepidermal elimination of thick coarse elastic fibres from the papillary dermis. The dermal infiltrate showed an immunohistological pattern consistent with an acute cell-mediated immune response. It consisted mainly of activated T-lymphocytes, with a predominance of CD4-positive cells. Considerable numbers of CD1-positive cells were also present. Inflammatory macrophages of the 27E10 phenotype were found in considerable numbers, whereas 25F9-positive resident macrophages were almost completely absent. The role of a cell-mediated immune response in the mechanism of transepithelial elimination is discussed.


Quantification of in vivo binding of [3H]RX 821002 in rat brain: evaluation as a radioligand for central alpha 2-adrenoceptors. On the basis of its established in vitro characteristics, [3H]RX 821002 was evaluated in rats as an in vivo radioligand for central alpha 2-adrenoceptors. Estimates for in vivo binding potential, obtained by compartmental analyses of time-radioactivity data, ranged between 1.9 for hypothalamus and 0.2 for cerebellum, with a regional distribution in brain which was similar to that observed in vitro. Selectivity and specificity of the signal were checked by predosing with either the alpha 2-antagonists, idazoxan or yohimbine, the alpha 2-agonist, clonidine, or the alpha 1-antagonist, prazosin. Pretreatment of the rats with the selective neurotoxin, DSP-4, had no significant effect on [3H]RX 821002 binding, suggesting that the majority of labelled sites were situated post-junctionally. The studies indicate that [3H]RX 821002 can be used experimentally as an in vivo marker for central alpha 2-adrenoceptors. The size and rate of expression of the specific signal encourage the development and assessment of [11C]RX 821002 for clinical PET studies.


[Chronic pancreatitis of the head of the pancreas in hyperplasia of the body and tail of the pancreas]. We report a case of chronic pancreatitis of the head of the pancreas complicated with insulin-requiring diabetes mellitus, in a 44-year-old woman with hypoplasia of the dorsal pancreas. Preoperative ultrasonography, computerized tomography and angiography revealed a calcifying retroperitoneal mass, which on explorative laparotomy proved to be a severe chronic pancreatitis of the head of the pancreas with a finding of abnormal visibility of the confluens venosum and absence of both the corpus and the tail of the pancreas. The postoperative course following pylorus-preserving duodenopancreatectomy was uneventful.


The evolution of proteinase substrates with special reference to dipeptidylpeptidase IV. The design and development of specific substrates for proteolytic enzymes is reviewed. Particular attention is given to substrates containing the leaving groups 4-methoxy-2-naphthylamide (MNA) and 7-amino-4-trifluoromethylcoumarin (AFC). The MNA substrates are used for histochemical and cytochemical purposes, and they yield a coloured final reaction product when azo-coupled with a diazonium salt, an osmiophilic product for electron microscopy when coupled with hexazotized Pararosaniline, or a fluorescent final reaction product when coupled with 5-nitrosalicylaldehyde. AFC substrates are considerably more sensitive, and they yield the fluorescent product AFC after enzymatic cleavage of the substrate. AFC is not sufficiently water-insoluble to allow (intra)cellular localization, but AFC substrates are successfully used for incubations in microwells (Immu-Probe technique) and for the demonstration of banding patterns after gel electrophoresis (enzyme-directed overlay membrane technique). The methods are discussed with the example of the elucidation of the role of dipeptidylpeptidase IV in autoimmune diseases.


Glutamate-like immunoreactivity in the leech central nervous system. Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.


Alloimmunization to platelet antigen HPA-1a (PIA1) is strongly associated with both HLA-DRB3*0101 and HLA-DQB1*0201. Antibodies to the platelet HPA-1a antigen can elicit in the newborn a condition known as neonatal alloimmune thrombocytopenic purpura (NAITP). Previous studies based on RFLP analysis showed that 100% of HPA-1a-negative women who produced anti-HPA-1a antibodies (responders) were HLA-DRw52a (DRB3*0101). However, this specificity could also be found in some HPA-1a-negative women not producing anti-HPA-1a antibodies (nonresponders). We have analyzed in detail by PCR-SSOP the HLA-DR, -DQ, and -DP loci of 36 responders and 10 nonresponders. We found that while the allele DRB3*0101 was present in the vast majority of responders (91%), there were exceptions. Furthermore, the DQB1*0201 allele was found to be present in almost all responders (94%), but again was also found in nonresponders. The risk of alloimmunization to HPA-1a in an HPA-1b homozygous mother significantly increases with the presence of either allele, the odds ratio being 39.7 for DQB1*0201 and 24.9 for DRB3*0101. Sequencing of exon 2 of these two alleles from responders indicated no sequence difference when compared with the consensus sequences. This indicates that they do not represent variants when compared with the same alleles found in some nonresponders.


Nucleotide sequences of the HLA-DRw12 and DRw8 B1 chains from an Australian aborigine. To gain a more detailed understanding of the molecular structure of the HLA genes in Australian aborigines, the polymorphic first-domain sequences of the DR B alleles were determined in an aborigine who was tissue typed as HLA-DRw8 and a probable DRw12; DRw52; DQw1,7. Both peripheral blood leukocytes and a lymphoblastoid cell line were reactive with the majority of DRw12-specific sera, but also with half of the DRw11-specific sera. With the use of primers specific for the conserved regions flanking the first domain, the polymerase chain reaction technique was used to amplify first-strand synthesis products prepared from the cell line. Two distinct DRB1 sequences were obtained. One was virtually identical to the reported DRw8,Dw8.3 sequence present in an Asian haplotype, differing only by a single silent nucleotide substitution at the third position of codon 36 (A to G). A second DRB allele was closely related to two recently published and nearly identical sequences for DRw12, with amino acid differences at positions 67 and 85 of the first domain. DRB RFLP studies on this cell line using the Taq I restriction enzyme indicated bands previously described for the DRw8 and DRw12 haplotypes.


Family study on HLA-DPB1 polymorphism: linkage analysis with HLA-DR/DQ and two "new" alleles. An extensive family study on HLA-DPB1 was performed in 105 families living in northeastern Japan. In a linkage study between HLA-DPB1 and other HLA loci, five apparent recombinations between DPB1 and DR/DQ loci were observed. The recombination frequency (theta) with maximum probability was estimated to be 0.017 by the lod score method. DPB1 allele and haplotype frequencies in unrelated parents were determined by direct counting. The most common allele was DPB1*0501 with the frequency of 41.2% and the second was DPB1*0201 with 24.0%. Nine DPB1-DR and six DPB1-DQ haplotypes were in significant linkage disequilibrium. Seven kinds of extended haplotypes were observed to be over 1%, in which the most common haplotype A24-B52-DR15-DQ6-DPB1*0901 occurred at 6.0%. Moreover, we found two "new" DPB1 alleles in this study. The first one possesses a single base substitution from DPB1*0501 resulting in an amino acid change. The other is most likely to be formed by an intraexonic recombination between DPB1*0301 and DPB1*0501.


A new perspective on stress ulcer prophylaxis. Gastric acid suppression by use of either antacids or histamine H2-receptor antagonist therapy is the mainstay of stress ulcer prophylaxis. Available evidence indicating an antimicrobial role for gastric acid calls for the reevaluation of gastric acid suppression. A pH of greater than 4.0 leads to bacterial overgrowth and colonization of the upper gastrointestinal tract which has been associated with nosocomial pneumonia, bacterial translocation from the gut, systemic sepsis, and multiple-organ failure. The availability of alternative therapy should discourage the routine use of acid-suppression therapy in the critically ill patient.


Implications of psychopharmacological studies for the practice of psychoanalysis. Over the past 35 years psychopharmacological developments have fueled major changes in the care of psychiatric patients. The most obvious of these are in two areas: (1) the control of symptoms not heretofore consistently treated effectively, such as the benefits of lithium in bipolar disorder, and (2) the shift toward reliance on medications rather than other forms of treatment. In this article I shall cover some of the implications of the second point, but will focus principally on other, less direct consequences of the development of specific pharmacotherapies for the practice of psychoanalysis. These areas are the enhanced importance of accurate diagnosis, the expansions of categories for which drugs are helpful, and the point, or framework, from which dynamic psychotherapies have their main indications.


Diagnostic efficacy of serum alkaline phosphatase and gamma-glutamyltransferase in dogs with histologically confirmed hepatobiliary disease: 270 cases (1980-1990). The diagnostic efficacy of serum alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) activities was examined, using the records of 270 dogs initially suspected of having hepatobiliary disease on the basis of history, findings on physical examination, results of baseline screening tests, or any combination of these data. Histologic examination of hepatic tissue was performed in each dog. Sixty-three dogs did not have histologic evidence of hepatobiliary disease and served as the control group. On the basis of diagnosis, dogs were assigned to 1 of 8 groups: dogs with cirrhosis (n = 34), steroid hepatopathy (n = 16), hepatic neoplasia (primary and secondary, n = 36), chronic hepatitis (n = 14), chronic passive congestion (n = 5), hepatic necrosis (n = 17), portosystemic vascular anomaly (n = 35), and cholestasis (extrahepatic bile-duct obstruction and intrahepatic cholestasis, n = 50). Of the 207 dogs with hepatobiliary disease, 29 (14%) had normal ALP and GGT activities, 31 (15%) had normal ALP activity, and 112 (54%) had normal GGT activity. Of the 63 control dogs, 29 (46%) had normal serum ALP and GGT activities, 32 had normal ALP activity (ALP specificity, 51%), and 55 had normal GGT activity (GGT specificity, 87%). The specificity of ALP and GGT in parallel (positive result = result of either test abnormal) was 46%, and in series (positive result = results of both tests abnormal) was 91%. The highest median activities of ALP developed in dogs with cholestasis, steroid hepatopathy, chronic hepatitis, and hepatic necrosis. The highest median activities of GGT developed in dogs with steroid hepatopathy, cholestasis, and hepatic necrosis.(ABSTRACT TRUNCATED AT 250 WORDS)


Construction of a cDNA library from microdissected guinea pig organ of Corti. Poly (A) RNA was isolated from microdissected guinea pig organ of Corti and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids were transformed into DH10B E. coli via electroporation. The library was found to have 3.35 x 10(6) independent colonies with ten percent of the colonies lacking an insert. After checking 33 randomly selected colonies for inserts, the average insert size was 1218 base pairs, ranging from 3300 base pairs to 400 base pairs. The library was screened with a beta-actin oligonucleotide probe and 1.4% of the colonies contained an insert hybridizing to the probe.


Effects of the beta-adrenergic agonist L644,969 on muscle protein turnover, endogenous proteinase activities, and meat tenderness in steers. Eight MARC III composite (1/4 Hereford, 1/4 Angus, 1/4 Pinzgauer, and 1/4 Red Poll) steers weighing approximately 350 kg were fed 0 or 3 ppm of the beta-adrenergic agonist L644,969 (Merck Sharp and Dohme Laboratories, Rahway, NJ) for 6 wk in a high-concentrate diet. Feed efficiency was higher (P less than .05) in beta-adrenergic agonist-fed steers at 1, 3, 5, and 6 wk on trial. Average daily gain was greater (P less than .05) in beta-adrenergic agonist-fed steers at 3, 5, and 6 wk on treatment. Fractional degradation rate (percentage/day) of skeletal muscle myofibrillar protein was 27.1% lower (P less than .05) in beta-adrenergic agonist-fed steers at 3 wk on trial. Fractional accretion rate (percentage/day) of skeletal muscle myofibrillar protein in beta-adrenergic agonist-fed steers was higher (P less than .05) at 1, 3, 5, and 6 wk on trial. The beta-adrenergic agonist-fed steers had heavier (P less than .05) carcasses (9.6%), larger (P less than .05) longissimus muscle areas (24.3%), and lower (P less than .05) USDA yield grades (43.8%). Marbling degree, USDA quality grade, kidney, pelvic, and heart fat percentage, and 12th rib fat thickness were not different (P greater than .05). Calpastatin activity was higher (P less than .05) in muscle from the beta-adrenergic agonist-fed steers at 0 and 7 d postmortem. There were no differences (P greater than .05) in mu- or m-calpain or in cathepsins B or B+L or cystatin(s) between beta-adrenergic agonist-fed and control steers.(ABSTRACT TRUNCATED AT 250 WORDS)


Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa. Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell.


The lethal phenotype caused by null mutations in the Escherichia coli htrB gene is suppressed by mutations in the accBC operon, encoding two subunits of acetyl coenzyme A carboxylase. Insertion mutations in the Escherichia coli htrB gene result in the unique phenotype of not affecting growth at temperatures below 32.5 degrees C but leading to a loss of viability at temperatures above this in rich media. When htrB bacteria growing in rich media were shifted to the nonpermissive temperature of 42 degrees C, they continued to grow at a rate similar to that at 30 degrees C but they produced phospholipids at the rate required for growth at 42 degrees C. This led to the accumulation of more than twice as much phospholipid per milligram of protein compared with that in wild-type bacteria. Consistent with HtrB playing a role in phospholipid biosynthesis, one complementation group of spontaneously arising mutations that suppressed htrB-induced lethality were mapped to the accBC operon. This operon codes for the biotin carboxyl carrier protein and biotin carboxylase subunits of the acetyl coenzyme A carboxylase enzyme complex, which catalyzes the first step in fatty acid biosynthesis. Four suppressor mutations mapped to this operon. Two alleles were identified as mutations in the accC gene, the third allele was identified as a mutation in the accB gene, and the fourth allele was shown to be an insertion of an IS1 transposable element in the promoter region of the operon, resulting in reduced transcription. The suppressor mutations caused a decrease in the rate of phospholipid biosynthesis, restoring the balance between the biosynthesis of phospholipids and growth rate, thus enabling htrB bacteria to grow at high temperatures.


Structural comparison of urease and a GroEL analog from Helicobacter pylori. Electron microscopy of purified protein preparations indicated that Helicobacter pylori urease consisted of circular particles that are 13 nm in diameter, some of which showed indications of threefold rotational symmetry. A GroEL analog of H. pylori (Hp60K) appeared as a disc-shaped molecule with a diameter similar to that of urease but possessed sevenfold rotational symmetry. In a side-view projection, Hp60K appeared as two or four discs stacked side by side.


Clozapine treatment of psychosis in Parkinson's disease: a report of five consecutive cases. Clozapine has gained acceptance as an antipsychotic in treatment-resistant schizophrenia. Its low propensity to induce extrapyramidal side effects makes clozapine an attractive treatment for patients with Parkinson's disease and dopaminomimetic psychosis. Recent evidence demonstrates that Parkinson's patients are exquisitely sensitive to both the antipsychotic and the potential extrapyramidal effects of clozapine. The uncontrolled studies suggest that low-dose clozapine may be efficacious in this population. The dose range, side effect profiles, and length of treatment varied in these reports. We report our experience with five patients with Parkinson's disease and psychosis who were treated with clozapine in an open trial. Three patients were successfully treated with clozapine (25-100 mg/day, mean = 66.7 mg) without worsening their parkinsonism. Adverse effects unrelated to the motor disability required discontinuation of clozapine in the other two patients. At 1- to 2-year follow-up, each patient had required increased dosages of clozapine (75-150 mg/day, mean = 125 mg) for continued management of their psychosis and parkinsonism. The higher dose range was well tolerated. These results suggest that clozapine may effectively treat psychosis in Parkinson's disease.


Reaction mechanism of glutamate racemase, a pyridoxal phosphate-independent amino acid racemase. Glutamate racemase of Pediococcus pentosaceus contained no cofactor, and was completely inactivated by a thiol reagent. The role of a cysteine residue in the enzyme reaction was studied by chemical modification. The modification of this cysteine residue resulted in a concomitant loss of activity. DL-Glutamate protected the enzyme from inactivation. The inactivated enzyme was reactivated by addition of dithiothreitol. The racemization in 2H2O showed an overshoot in the optical rotation of glutamate before the substrate was completely racemized. This indicates that the removal of alpha-hydrogen is the rate determining step. During the racemization of D- or L-glutamate in 3H2O, tritium was incorporated preferentially into the product. Glutamate is racemized by the enzyme probably through a two base mechanism.


Rat liver dipeptidylpeptidase IV contains competing apical and basolateral targeting information. Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV (DPPIV). Most newly synthesized rat liver DPPIV is delivered directly to the apical surface of transfected MDCK cells; however, about 20% is delivered first to the basolateral surface and reaches the apical surface via transcytosis (Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) J. Biol. Chem. 266, 24428-24432). A soluble form of DPPIV (solDPPIV) containing only the lumenal domain of the protein was efficiently transported and secreted by stably transfected MDCK cells. If this domain contains apical sorting information, we would expect 80% of the soluble protein to be secreted apically. Surprisingly, 95% of the secreted solDPPIV was found in the apical medium. The high efficiency of apical secretion suggested that the transmembrane domain and cytoplasmic tail of DPPIV might contain competing basolateral targeting information. To test this hypothesis, we investigated the trafficking of a chimera in which the cytoplasmic tail and transmembrane domains of DPPIV were joined to lysozyme, an exogenous protein which should not contain sorting information. This protein was delivered predominantly to the basolateral surface. Our results suggest that the lumenal domain of DPPIV carries dominant apical sorting information while the transmembrane domain and cytoplasmic tail of the molecule contains competing basolateral sorting information.


Activation of Ca2+/calmodulin-dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons. In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) through autophosphorylation when the neurons were incubated in Mg(2+)-free buffer, and this response was blocked by specific antagonists of the N-methyl-D-aspartate (NMDA) receptor. In addition, glutamate stimulated the transient translocation of protein kinase C (PKC) from the cytosol to the membrane fraction. This effect was not blocked by NMDA receptor antagonists but was partially blocked by DL-2-amino-3-phosphonopropionate. Quisqualate or trans-1-amoinocyclopentane-trans1,3-dicarboxylate produced a similar effect on the translocation of PKC. In the experiments with 32P-labeled cells, the phosphorylation of microtuble-associated protein 2 and synapsin I, as well as autophosphorylation of CaM kinase II, were found to be stimulated by exposure to glutamate. These results suggest that glutamate can activate CaM kinase II through the ionotropic NMDA receptor, which in turn increases the phosphorylation of microtuble-associated protein 2 and synapsin I. PKC was activated through the metabotropic glutamate receptor in the hippocampal neurons.