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Biomedical subjects

D Klein

Publications and source records attributed to D Klein.

At least 19 recordsLinked to original sources
PubMed1992

Major histocompatibility complex class II genes of zebrafish.

Twenty cDNA clones derived from beta-chain-encoding class II genes of the zebrafish (Brachydanio rerio) major histocompatibility complex (MHC) have been sequenced. They fall into three groups identifying three loci of expressed genes. The length and organization of these genes are similar to those of their mammalian homologs. Amplification by polymerase chain reaction and sequencing of genomic DNA from zebrafish collected at different locations in India indicate the existence of a fourth group of sequences (fourth locus). A high degree of polymorphism at the B. rerio MHC loci and concentration of variability to the putative peptide-binding region of the beta 1-domain-encoding part of the gene are also indicated. Large genetic distances between alleles suggest trans-specific evolution of fish MHC polymorphism. Zebrafish genes appear to be derived from a different ancestor than the various class II gene families of other vertebrates. In spite of great sequence divergence between fish and mammalian MHC genes, there seems to be a striking conservation in their overall organization.

Animals↗
PubMed1992

Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases.

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65----Arg mutant with the peptide and thiolester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20,000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58----Leu, Tyr90----Asn, Thr101----Asn, Phe164----Ala, Asp225----Glu and Asp225----Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164----Ala mutant was significantly more unstable than the wild-type enzyme.

Amino Acid Sequence↗
PubMed1992

The evolutionary origin of the HLA-DR3 haplotype.

The human HLA-DR3 haplotype consists of two functional genes (DRB1*03 and DRB3*01) and one pseudogene (DRB2), arranged in the order DRB1...DRB2...DRB3 on the chromosome. To shed light on the origin of the haplotype, we sequenced 1480 nucleotides of the HLA-DRB2 gene and long stretches of two other genes, Gogo-DRB2 from a gorilla, "Sylvia" and Patr-DRB2 from a chimpanzee, "Hugo". All three sequences (HLA-DRB2, Gogo-DRB2, Patr-DRB2) are pseudogenes. The HLA-DRB2 and Gogo-DRB2 pseudogenes lack exon 2 and contain a twenty-nucleotide deletion in exon 3, which destroys the correct translational reading frame and obliterates the highly conserved cysteine residue at position 173. The Patr-DRB2 pseudogene lacks exons 1 and 2; it does not contain the twenty-nucleotide deletion, but does contain a characteristic duplication of that part of exon 6 which codes for the last four amino acid residues of the cytoplasmic region. When the nucleotide sequences of these three genes are compared to those of all other known DRB genes, the HLA-DRB2 is seen as most closely related to Gogo-DRB2, indicating orthologous relationship between the two sequences. The Patr-DRB2 gene is more distantly related to these two DRB2 genes and whether it is orthologous to them is uncertain. The three genes are in turn most closely related to HLA-DRBVI (the pseudogene of the DR2 haplotype) and Patr-DRB6 (another pseudogene of the Hugo haplotype), followed by HLA-DRB4 (the functional but nonpolymorphic gene of the DR4 haplotype). These relationships suggest that these six genes evolved from a common ancestor which existed before the separation of the human, gorilla, and chimpanzee lineages. The DRB2 and DRB6 have apparently been pseudogenes for at least six million years (myr). In the human and the gorilla haplotype, the DRB2 pseudogene is flanked on each side by what appear to be related genes. Apparently, the DR3 haplotype has existed in its present form for more than six myr.

Animals↗
PubMed1992

Trans-species evolution of Mhc-DRB haplotype polymorphism in primates: organization of DRB genes in the chimpanzee.

The DRB region of the human major histocompatibility complex displays length polymorphism: Five major haplotypes differing in the number and type of genes they contain have been identified, each at appreciable frequency. In an attempt to determine whether this haplotype polymorphism, like the allelic polymorphism, predates the divergence of humans from great apes, we have worked out the organization of the DRB region of the chimpanzee Hugo using a combination of chromosome walking, pulsed-field gel electrophoresis, and sequencing. Hugo is a DRB homozygote whose single DRB haplotype is some 440 kilobases (kb) long and contains five genes. At least one and possibly two of these are pseudogenes, while three are presumably active genes. The genes are designated DRB*A0201, DRB2*0101, DRB3*0201, DRB6*0105, and DRB5*0301, and are arranged in this order on the chromosome. The DRB2 and DRB3 genes are separated by approximately 250 kb of sequence that does not seem to contain any additional DRB genes. The DRB*A0201 gene is related to the DRB1 gene of the human DR2 haplotype; the DRB2*0101 and DRB3*0201 genes are related to the DRB2 and DRB3 genes of the human DR3 haplotype, respectively; the DRB6*0105 and DRB5*0301 genes are related to the DRBVI and DRB5 genes of the human DR2 haplotype, respectively. Thus the Hugo haplotype appears to correspond to the entire human DR2 haplotype, into which a region representing a portion of the human DR3 haplotype has been inserted. Since other chimpanzees have their DRB regions organized in different ways, we conclude that, first, the chimpanzee DRB region, like the human DRB region, displays length polymorphism; second, some chimpanzee DRB haplotypes are longer than the longest known human DRB haplotypes; third, in some chimpanzee haplotypes at least, the DRB genes occur in combinations different from those of the human haplotypes; fourth, and most importantly, certain DRB gene combinations have been conserved in the evolution of chimpanzees and humans from their common ancestors. These data thus provide evidence that not only allelic but also haplotype polymorphism can be passed on from one species to another in a given evolutionary lineage.

Animals↗
PubMed1992

Shared polymorphism between gorilla and human major histocompatibility complex DRB loci.

A high degree of polymorphism and high nucleotide diversity mark the functional genes of the major histocompatibility complex (Mbc). Alleles at the different Mbc loci can be classified into distinct lineages that are shared between species and, therefore, are presumed to have been founded before speciation. We have sequenced the most polymorphic part of 25 gorilla Mbc-DRB genes from six individuals. (The DRB genes code for the beta-polypeptide chain of the alpha beta heterodimer that constitutes one family of the class II MHC molecules.) Fifteen of the sequences identify new alleles at four DRB loci; each of the six gorillas was heterozygous at one of the loci at least. Thirteen of the alleles could be assigned to lineages identified previously; the remaining two alleles represent new lineages. All the major human DRB allelic lineages are now known to be shared with apes, and all must have originated before the human-gorilla-chimpanzee divergence more than six million years (my) ago. The presence of some of the gorilla and human lineages in Old World monkeys suggests that these lineages emerged before the divergence of apes and cercopithecids. We argue that the major allelic lineages at the DRB1 locus began to diverge shortly after the rounds of duplication that generated the different DRB loci now found in the hominoids and that this event occurred more than 30 my ago. Comparison of closely related gorilla DRB sequences indicates that polymorphism may be generated by several mechanisms: point mutations, slippage during DNA replication, and recombination. Deduced gene linkages provide evidence for transspecies evolution of haplotype polymorphism.

Alleles↗
PubMed1992

Comparative anatomy of the primate major histocompatibility complex DR subregion: evidence for combinations of DRB genes conserved across species.

The class II region of the human major histocompatibility complex (HLA) is made up of three major subregions designated DR, DQ, and DP. With the aim of gaining an insight into the evolution and stability of DR haplotypes, a total of 63 cosmid clones were isolated from the DR subregion (Gogo-DR) of a western lowland gorilla. All but one of these cosmid clones were found to fall into two clusters. The larger cluster, A, was defined by 41 overlapping cosmid clones and contained a DRB gene segment made up of exons 4 through 6 and four DRB genes, designated Gogo-DRB6, Gogo-DRB5*01, Gogo-DRB8, and Gogo-DRB3*01. The total length of this cluster was approximately 180 kb. The second cluster, B, encompassed a contiguous DNA stretch of approximately 145 kb and was composed of 21 overlapping cosmid clones. Cluster B contained three DRB genes, designated Gogo-DRB1*08, Gogo-DRB2, and Gogo-DRB3*02. One cosmid clone (WP1-9) containing a DRB pseudogene could not be linked to either cluster A or B. Neither the organization of cluster A nor that of cluster B was identical to that of known HLA-DR haplotypes. However, two gorilla DRB genes, Gogo-DRB6 and Gogo-DRB5*01, the human counterparts of which are linked in the HLA-DR2 haplotype, were found to be located next to each other in cluster A. The arrangement of the Gogo-DRB genes in cluster B, which is presumed to be the gorilla DR8 haplotype, was similar to that of HLA-DR3/DR5/DR6 haplotypes and to that of the presumed ancestral HLA-DR8 haplotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Ciprofloxacin in the treatment of Mycobacterium fortuitum infection of the peroneal tendons. A case report.

In the case reported, M. fortuitum was sensitive in vitro to amikacin, erythromycin, tobramycin, and ciprofloxacin. Because the patient did not respond to long-term therapy with amikacin and erythromycin, an experimental antibiotic, ciprofloxacin, was tried. Only after extensive surgical debridement and 2 1/2 months of oral ciprofloxacin therapy was the infection eradicated and wound healing obtained. The authors conclude that a wound that has reopened, but remains indolent, exudes a clear, serous drainage and responds poorly to antibiotics should suggest a possible mycobacterial infection. Combination antibiotic therapy is recommended because of the high rate of relapse and development of resistance to drugs. Extensive surgical debridement of all infected tissue remains the primary treatment. The therapeutic value of ciprofloxacin and other newer antibiotics in the treatment of mycobacterial infection is promising.

Adult↗
PubMed1992

[Treatment of Fournier's gangrene. A review based on 3 new cases].

We describe three new cases of Fournier's gangrene-a necrotizing fasciitis of urogenital or anorectal origin. Though in the initial report the disease was believed to be idiopathic, the source of infection or immuncompromising factors can be identified in nearly all cases today. We present a combination of aggressive surgical therapy and adjunctive use of Imipenem which was successful in the treatment of all our cases. By using fully resorbable nutrition colostomy could be avoided successfully.

Adult↗
PubMed1992

Molecular genetic study of human arginase deficiency.

We have explored the molecular pathology in 28 individuals homozygous or heterozygous for liver arginase deficiency (hyperargininemia) by a combination of Southern analysis, western blotting, DNA sequencing, and PCR. This cohort represents the majority of arginase-deficient individuals worldwide. Only 2 of 15 homozygous patients on whom red blood cells were available had antigenically cross-reacting material as ascertained by western blot analysis using anti-liver arginase antibody. Southern blots of patient genomic DNAs, cut with a variety of restriction enzymes and probed with a near-full-length (1,450-bp) human liver arginase cDNA clone, detected no gross gene deletions. Loss of a TaqI cleavage site was identified in three individuals: in a homozygous state in a Saudi Arabian patient at one site, at a different site in homozygosity in a German patient, and in heterozygosity in a patient from Australia. The changes in the latter two were localized to exon 8, through amplification of this region by PCR and electrophoretic analysis of the amplified fragment after treatment with TaqI; the precise base changes (Arg291X and Thr290Ser) were confirmed by sequencing. It is interesting that the latter nucleotide variant (Thr290Ser) was found to lie adjacent to the TaqI site rather than within it, though whether such a conservative amino acid substitution represents a true pathologic mutation remains to be determined. We conclude that arginase deficiency, though rare, is a heterogeneous disorder at the genotypic level, generally encompassing a variety of point mutations rather than substantial structural gene deletions.

Amino Acid Sequence↗
PubMed1991

The mutation Lys234His yields a class A beta-lactamase with a novel pH-dependence.

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

Ampicillin↗
PubMed1991

Effect of an adjacent base on detection of a point mutation by restriction enzyme digestion.

While routinely mapping point mutations within the arginase locus of a collection of hyperargininemic patients, we discovered that a base immediately outside a restriction endonuclease recognition site (TaqI) can eliminate cleavage of this site by this enzyme. The genetic lesion lay in a base immediately flanking a TaqI recognition site within exon 8 of the arginase locus and abolished cutting by approximately 80%. We wish to emphasize the necessity of heeding subtle cues frequently encountered while generating restriction enzyme data, because neither Southern blot maps nor endonuclease digestion of polymerase chain reaction amplified products of exon 8 accurately predicted where the point mutation lay. To our knowledge, this is the first instance of inhibition of cleavage by flanking bases occurring on natural (nonsynthetic) DNA substrates, i.e., within the clinical setting of characterization of a human genetic disorder.

Base Sequence↗
PubMed1991

Gorilla major histocompatibility complex-DRB pseudogene orthologous to HLA-DRBVIII.

The HLA-DR4 haplotype consists of four DRB genes: DRB1*04, DRBVII, DRBVIII, and DRB4*01, arranged in this order on the chromosome. The DRB1 and DRB4 genes code for beta chains of the alpha beta heterodimers expressed on the cell surface and bearing the HLA-DR4 and HLA-DRw53 determinants, respectively; the DRBVII and DRBVIII are pseudogenes. We found and sequenced a gene closely related to HLA-DRBVIII in the genome of the lowland gorilla "Sylvia." We designate this gene Gogo-DRB8. The close relationship between the two genes is indicated by the overall sequence similarity, the absence of recognizable exons 1 and 2 in both genes, the presence of two Alu repeats at corresponding positions, and high sequence similarity between corresponding repeats. The comparison with an outgroup (tamarin) gene and the functional counterparts of the DRB8 gene indicate that DRB8 emerged between 18 and 26 million years ago and became inactivated at the same time as or shortly after its creation. Hence DRB8 has probably existed as a pseudogene since the divergence of apes from Old World monkeys more than 20 million years ago.

Animals↗
PubMed1991

Metallothionein, copper and zinc in fetal and neonatal human liver: changes during development.

Total and cytosolic zinc (Zn) and copper (Cu), cytosolic metallothionein (MT) and the Cu-load of MT were investigated in fetal (22, 24 and 32 gestational weeks) and neonatal (2-15 months) human liver. Whereas the fraction of cytosolic Zn remained constant at 66% of the total independent of the stage of development, the fraction of cytosolic Cu increased from 26% in preterm liver to about 100% within 12 months postnatally. The MT content was higher in fetal than in neonatal liver. There was a linear correlation (r = 0.996) between cytosolic MT and Zn in both fetal and neonatal liver but not between MT and Cu. In contrast to fetal liver, the Cu-load of MT in neonatal liver seems to be determined by the Zn/Cu ratio in the cytosol. The results suggest that MT is involved in the regulation of Cu and Zn metabolism during fetal and neonatal development.

Aging↗