Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8-A resolution

It has recently been demonstrated that the C-terminal deletion mutant of recombinant human carbonic anhydrase IV (G267X CA IV) converts the normally glycosylphosphatidylinositol-anchored enzyme into a soluble secretory form which has the same catalytic properties as the membrane-associated enzyme purified from human tissues. We have determined the three-dimensional structure of the secretory form of human CA IV by x-ray crystallographic methods to a resolution of 2.8 A. Although the zinc binding site and the hydrophobic substrate binding pocket of CA IV are generally similar to those of other mammalian isozymes, unique structural differences are found elsewhere in the active site. Two disufide linkages, Cys-6-Cys-11G and Cys-23-Cys-203, stabilize the conformation of the N-terminal domain. The latter disulfide additionally stabilizes an active site loop containing a cis-peptide linkage between Pro-201 and Thr-202 (this loop contains catalytic residue Thr-199). On the opposite side of the active site, the Val-131-Asp-136 segment adopts an extended loop conformation instead of an alpha-helix conformation as found in other isozymes. Finally, the C terminus is surrounded by a substantial electropositive surface potential, which is likely to stabilize the interaction of CA IV with the negatively charged phospholipid headgroups of the membrane. These structural features are unique to CA IV and provide a framework for the design of sulfonamide inhibitors selective for this particular isozyme.     

Characterization of a subunit structure and stability of the recombinant porin from Neisseria gonorrhoeae

An outer membrane PIA protein from Neisseria gonorrhoeae strain FA19 was expressed in Escherichia coli and refolded in vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50 degrees C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80 degrees C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind with Kd=80 and 130 microM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.     

Dorzolamide: development and clinical application of a topical carbonic anhydrase inhibitor

Systemic carbonic anhydrase inhibitors are among the most powerful agents to lower intraocular pressure. Unfortunately, their use is frequently accompanied by undesired side effects. Some are due to the relatively large amounts of drug that have to be systemically administered to inhibit the carbonic anhydrase in the ciliary processes. Recently, dorzolamide, a topical carbonic anhydrase inhibitor, has become commercially available for clinical use. This article reviews the development of topical carbonic anhydrase inhibitors with special reference to dorzolamide. When administered three-times daily, dorzolamide lowers intraocular pressure in a clinically useful manner. Ocular side-effects include frequent stinging and burning and allergy can develop. Systemic side effects have not been observed that could definitively be related to inhibition of extraocular carbonic anhydrase. Blood dyscrasias have not yet been observed. Absence of cardiovascular and pulmonary side effects, as can occur with beta adrenergic antagonists, and lack of pupillary and accommodative stimulation, as occur with cholinergic agonists, might make dorzolamide first-line medical treatment for elevated intraocular pressure.     

Crystal structure of a bacterial signal peptidase in complex with a beta-lactam inhibitor

The signal peptidase (SPase) from Escherichia coli is a membrane-bound endopeptidase with two amino-terminal transmembrane segments and a carboxy-terminal catalytic region which resides in the periplasmic space. SPase functions to release proteins that have been translocated into the inner membrane from the cell interior, by cleaving off their signal peptides. We report here the X-ray crystal structure of a catalytically active soluble fragment of E. coli SPase (SPase delta2-75). We have determined this structure at 1.9 A resolution in a complex with an inhibitor, a beta-lactam (5S,6S penem), which is covalently bound as an acyl-enzyme intermediate to the gamma-oxygen of a serine residue at position 90, demonstrating that this residue acts as the nucleophile in the hydrolytic mechanism of signal-peptide cleavage. The structure is consistent with the use by SPase of Lys 145 as a general base in the activation of the nucleophilic Ser90, explains the specificity requirement at the signal-peptide cleavage site, and reveals a large exposed hydrophobic surface which could be a site for an intimate association with the membrane. As enzymes that are essential for cell viability, bacterial SPases present a feasible antibacterial target: our determination of the SPase structure therefore provides a template for the rational design of antibiotic compounds.     

Effects of a carbonic anhydrase inhibitor on cerebral blood flow in geriatric patients

A carbonic anhydrase inhibitor (UK-12,130) was shown to increase cerebral blood flow in mildly demented geriatric patients. Oral administration caused a significant increase in blood flow at two different dose levels; this persisted for at least six weeks, which was the duration of the longest study. There was no consistent improvement in mentation during treatment. Blood blow was measured by the washout of 133Xe after inhalation of this inert gas.     

Characteristics of atypical Neisseria gonorrhoeae from disseminated and localized infections

Approximately 6% of 1,200 clinical isolates of Neisseria gonorrhoeae were atypical because they produced smaller than normal colonies on conventioal chocolate agar and fermented glucose weakly. Auxotyping studies indicated that these atypical strains required for growth arginine, uracil, and, in most instances, hypoxanthine. In addition, all of them were susceptible to 0.02 U of penicillin/ml. None of the normal colony isolates, including those susceptible to the same low concentration of penicillin, had the same nutritional characteristics. Atypical strains comprised almost half of the isolates from disseminated infections, but only 5% of those from localized infections. Auxotyping was used to identify the contact of a patient who became reinfected nine times with an atypical gonoccal strain. In addition to its usefulness in such epidemiological studies, this technique has enabled us to distinguish a subgroup of gonococci with apparent increased pathogenicity.     

Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.     

Alpha-crystallin, a molecular chaperone, forms a stable complex with carbonic anhydrase upon heat denaturation

Alpha-crystallin, a major eye lens protein of vertebrates has been characterized as a molecular chaperone based on its ability to inhibit the aggregation of proteins undergoing thermal denaturation (Horwitz, J., Proc. Natl. Acad. Sci. USA 1992, 89, 10449-10453). To understand the mechanisms underlying this chaperone-like activity, the present study addressed molecular interactions between alpha-crystallin and its target proteins. Using carbonic anhydrase as a model target protein, we demonstrate complex formation between the 2 proteins upon heating, as assessed by the criteria of agarose gel electrophoresis, immunoprecipitation, ultrafiltration and gel filtration chromatography. The complex of alpha-crystallin and carbonic anhydrase is stable, at room temperature and at 4 degrees C, for over 18 hours, and is non-covalent in nature. The results also indicate that alpha-crystallin binds the early non-native form of the target protein.     

Clinical use of a topical carbonic anhydrase inhibitor in patients affected by chronic simple glaucoma

The aim of this study is to evaluate the ocular hypotensive efficacy of a topical carbonic anhydrase inhibitor (dorzolamide) in primary open-angle glaucoma patients, administered alone or in association with beta-blockers or with beta-blockers and miotics, in a one-year follow-up.     

Isolation and expression of murine carbonic anhydrase IV

Many studies have demonstrated the problems patients have with medications. In these studies, the elderly and patients utilizing psychiatric medications are often cited as having the greatest noncompliance. This article reviews the extent of noncompliance in this population and the effect of noncompliance on disease management. Factors related to the health care provider, the patient, and the type of therapy that lead to noncompliance are reviewed. Opportunities and intervention techniques are provided to improve patient outcomes.     

Identification and characterization of human neutrophil carbonic anhydrase

The transition into rehabilitative treatment following detoxification is a critical event for most chemically dependent individuals. A host of factors may enhance or impede this transition, yet little or no research has been done to shed light on this issue. This paper presents the results of the fortuitous changing of one variable which appeared to increase the likelihood of a successful transition, namely, the degree of physical proximity of the detox program and the rehab program. Results indicated a significantly greater proportion of patients successfully making the transition from detox to rehab when the programs were very close proximally than when they were less close. The role of possible patient-to-patient modeling effects was discussed.     

Cloning and characterization of the gene encoding a new DNA methyltransferase from Neisseria gonorrhoeae

A HindIII fragment of N. gonorrhoeae MS11 DNA coding for DNA methyltransferase (MTase) activity was cloned and expressed in E. coli AP1-200-9 cells. The sequence of 4681 bp was determined, and its analysis revealed two open reading frames (ORFs) sharing some similarity with known DNA MTases. ORF1 encodes an active N4mC MTase (M.NgoMV). The enzyme modifies only one strand of double stranded DNA and preferentially recognises the sequence GCCHR although it is able to methylate other sites. The exact recognition sequence cannot be precisely defined due to a relaxed specificity. The second ORF shows high homology to 5mC Mtases, but we were unable to demonstrate DNA methylating activity of its product either in vivo or in vitro.     

Chromatographic and electrophoretic methods related to the carbonic anhydrase isozymes

There are three gene families that encode zinc metalloenzymes that catalyze the reversible hydration of CO2. The encoded enzymes are termed carbonic anhydrases (CAs). The CA isozymes have been purified from representatives of all types of organisms. Most CAs are strongly inhibited by aromatic sulfonamides. Several chromatographic and electrophoretic methods have been devised to determine binding constants for sulfonamides to CAs, and these compounds have been extensively used for, often single-step, affinity chromatographic separation of CAs from complex matrixes. The purification of different CA isozymes from different organisms is reviewed, as are methods for detection of CAs during chromatography and electrophoresis.     

Carbonate dehydratase (carbonic anhydrase) and the fetal lung

Carbonic anhydrase activity (carbonic dehydratase, EC 4.2.1.1) has been detected in the fetal lungs of stillborn human infants and rhesus monkeys, but a role for this enzyme in the fetal lung has not been elucidated. In utero the mammalian lung develops as a liquid-filled structure, the liquid being secreted by the lung. In the fetal lamb this liquid, when compared with plasma, has a high chloride and a low bicarbonate concentration, suggesting a possible role for carbonate dehydratase. Studies on 10 fetal lambs confirmed the presence of carbonate dehydratase in the lung. Levels at 60-66 days were negligible and rose to 0.30 Meldrum Roughton units/mg protein at about 140 days (term 147 days), with little change after birth. In another six fetal lambs at 135-136 days, inhibition of this enzyme with 100 mg acetazolamide suppressed the mean rate of secretion of lung liquid by 64.5% (P less than 0.005), which correlated with a significant drop in chloride concentration (P less than 0.001). This magnitude of changes in secretion after acetazolamide is of the same order as that occurring in the secretion of cerebrospinal fluid when carbonate dehydratase is inhibited. This observation supports the hypothesis that carbonate dehydratase in fetal lung affects the secretion of lung liquid, although its mechanism is as yet unknown.     

Isolation and characterization of a carbonic anhydrase homologue from the zebrafish (Danio rerio)

Lymphomas with T-cell phenotype represent a heterogeneous group of diseases differing in histopathology, tumour site, and cell origin. They include peripheral T-cell lymphomas (PTCLs) derived from alpha beta cells, but also some recently recognized entities such as gamma delta, hepatosplenic lymphomas and natural killer (NK) cell lymphomas. Only a few studies have investigated the possibility that at least some PTCLs could be derived from lymphocytes with cytotoxic potential. In order to investigate this possibility, 60 cases of PTCL, including 27 cases expressing the alpha beta T-cell receptor (TCR alpha beta), 15 TCR gamma delta cases and 18 cases expressing neither TCR (TCR silent), as well as 14 cases of NK-cell lymphomas, were studied by immunohistochemistry for the expression of TIA-1, perforin, and granzyme B proteins. Expression of TIA-1 is characteristic of cytotoxic cells regardless of their activation status, whereas expression of perforin and granzymes is highly increased in activated cytotoxic cells and correlates with the induction of cytolytic activity. All NK-cell lymphomas (11 sinonasal, three systemic cases) expressed TIA-1, perforin, and granzyme B in most tumour cells. All gamma delta PTCLs (15 cases) expressed TIA-1 protein in most tumour cells, with a different cytotoxic antigen profile in hepatosplenic gamma delta PTCL (TIA-1+, perforin-, granzyme B-) and in non-hepatosplenic gamma delta PTCLs (three nasal, one skin, one lung), the latter expressing the three cytotoxic proteins. Of the 45 cases of alpha beta and TCR silent PTCL, 15 (33 per cent) were considered to be derived from cytotoxic lymphocytes with expression of at least one cytotoxic protein (TIA-1, 15/45; perforin, 10/41; granzyme B, 14/38) in tumour cells. This cytotoxic protein expression appeared to be related to the site of localization, since 7/13 (54 per cent) extranodal and only 8/32 (25 per cent) nodal alpha beta and TCR silent PTCLs expressed TIA-1, and to histology, since this pattern was observed in a proportion of anaplastic (6/8, 75 per cent) and pleomorphic (8/17, 47 per cent) lymphomas, but not in AILD-type NHL (0/16). Taken together, our data suggest that NK-cell lymphomas and non-hepatosplenic gamma delta PTCLs represent tumours of activated cytotoxic NK cells and gamma delta T cells, respectively; that hepatosplenic gamma delta PTCLs represent tumours of non-activated cytotoxic gamma delta T cells; and that a small proportion of alpha beta and TCR silent PTCLs, mostly extranodal cases, or nodal anaplastic lymphomas, represent tumours of cytotoxic T cells.     

Antibiotic susceptibility of Neisseria gonorrhoeae in Trinidad and Tobago

The authors studied the state of humoral immunity in thirty six female individuals with normal pregnancy, 52 ones presenting with mild anemia, 33 with moderately severe, and 14 with severe anemia, as well as in twelve healthy non-pregnant women. The studies were made by trimesters in the time course of pregnancy. Those pregnant with no anemia demonstrated lowering of B-lymphocytes, rise in circulating immune complexes (CIC) as compared to the non-pregnant individuals; no difference in the immunoglobulins content was noted. In anemia of the pregnant women the immunity B-system gets suppressed with progression of anemia, CIG tend to be on the increase, IgM and IgG get augmented during the second and third trimesters of pregnancy. The above changes suggest some inadequacy of humoral immunity in anemia of pregnancy.     

Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family

The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family. We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library. Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column. Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved. However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin. pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding. In addition, the antigenic determinants of pICA and the transferrins are not identical. These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.     

Identification and functional characterization of the Neisseria gonorrhoeae lbpB gene product

We cloned lbpB, encoding a predicted 80-kDa lipoprotein, upstream of lbpA. A nonpolar mutant (LbpB- LbpA+) had normal lactoferrin (LF) binding and grew normally with LF as an iron source, whereas LbpB- LbpA- and LbpB+ LbpA- strains had reduced binding of LF and did not grow with LF as an iron source. LbpB bound LF directly in an affinity purification, suggesting that LbpB might play a still-uncharacterized role in the LF iron utilization.