Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12.

A new gene, designated rcsF, was located adjacent to drpA at the 5.2-min position of the genetic map of Escherichia coli. The deduced amino acid sequence encoded by the rcsF gene indicates a small protein of 133 amino acid residues with a calculated pI of 10.8 that is rich in proline, serine, alanine, and cysteine residues. When overexpressed as a result of its presence on a multicopy plasmid, rcsF confers a mucoid phenotype and restores colony formation to ftsZ84 mutant cells on L agar medium containing no added NaCl. These two phenotypes are not observed in rcsB mutant cells. Ion mutant cells harboring an rcsF mutation accumulate considerably lower levels of exopolysaccharides, whereas the presence of a multicopy rcsF plasmid not only increases capsule synthesis but also confers a mucoid phenotype at 37 degrees C, a temperature at which ion mutant cells are known not to form mucoid colonies. RcsF does not stimulate the expression of rcsB, indicating that it exerts its action through the RcsB protein, possibly by phosphorylation. It is also shown that RcsF stimulation of capsule synthesis is RcsA-dependent, whereas colony formation of ftsZ84 mutant cells can be restored by RcsF in the absence of RcsA.


Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.


Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli.

Fis is a small basic DNA-binding protein from Escherichia coli that was identified because of its role in site-specific DNA recombination reactions. Recent evidence indicates that Fis also participates in essential cell processes such as rRNA and tRNA transcription and chromosomal DNA replication. In this report, we show that Fis levels vary dramatically during the course of cell growth and in response to changing environmental conditions. When stationary-phase cells are subcultured into a rich medium, Fis levels increase from less than 100 to over 50,000 copies per cell prior to the first cell division. As cells enter exponential growth, nascent synthesis is largely shut off, and intracellular Fis levels decrease as a function of cell division. Fis synthesis also transiently increases when exponentially growing cells are shifted to a richer medium. The magnitude of the peak of Fis synthesis appears to reflect the extent of the nutritional upshift. fis mRNA levels closely resemble the protein expression pattern, suggesting that regulation occurs largely at the transcriptional level. Two RNA polymerase-binding sites and at least six high-affinity Fis-binding sites are present in the fis promoter region. We show that expression of the fis operon is negatively regulated by Fis in vivo and that purified Fis can prevent stable complex formation by RNA polymerase at the fis promoter in vitro. However, autoregulation only partially accounts for the expression pattern of Fis. We suggest that the fluctuations in Fis levels may serve as an early signal of a nutritional upshift and may be important in the physiological roles Fis plays in the cell.


Role of PhoU in phosphate transport and alkaline phosphatase regulation.

The negative regulatory function of PhoU in alkaline phosphatase (AP) was suggested by the behavior of K10 phoU35 carrying a missense mutation whose product was detected by immunoblotting. To define more clearly the regulatory function of this protein for the synthesis of AP, we constructed a null mutation. The constitutive synthesis of AP in this phoU deletion strain confirmed the negative role of PhoU. However, the expression of the PhoU protein from an isopropyl-beta-D-thiogalactopyranoside-inducible promoter had no effect on the repression of AP synthesis. Furthermore, the involvement of PhoU in free-Pi uptake was demonstrated. These results provide evidence that PhoU participates in Pi transport and in the regulatory role of the phosphate-specific transport system.


Lysis of lysis-inhibited bacteriophage T4-infected cells.

T4 bacteriophage (phage)-infected cells show a marked increase in latent-period length, called lysis inhibition, upon adsorption of additional T4 phages (secondary adsorption). Lysis inhibition is a complex phenotype requiring the activity of at least six T4 genes. Two basic mysteries surround our understanding of the expression of lysis inhibition: (i) the mechanism of initiation (i.e., how secondary adsorption leads to the expression of lysis inhibition) and (ii) the mechanism of lysis (i.e., how this signal not to lyse is reversed). This study first covers the basic biology of the expression of lysis inhibition and lysis of T4-infected cells at high culture densities. Then evidence is presented which implies that, as with the initiation of lysis inhibition, sudden, lysis-associated clearing of these cultures is likely caused by T4 secondary adsorption. For example, such clearing is often observed for lysis-inhibited T4-infected cells grown in batch culture during T4 stock preparation. The significance of this secondary adsorption-induced lysis to wild T4 populations is discussed. The study concludes with a logical argument suggesting that the lytic nature of the T4 phage particle evolved as a novel mechanism of phage-induced lysis.


Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum.

Threonine dehydratase activity is an important element in the flux control of isoleucine biosynthesis. The enzyme of Corynebacterium glutamicum demonstrates a marked sigmoidal dependence of initial velocity on the threonine concentration, a dependence that is consistent with substrate-promoted conversion of the enzyme from a low-activity to a high-activity conformation. In the presence of the negative allosteric effector isoleucine, the K0.5 increased from 21 to 78 mM and the cooperativity, as expressed by the Hill coefficient increased from 2.4 to 3.7. Valine promoted opposite effects: the K0.5 was reduced to 12 mM, and the enzyme exhibited almost no cooperativity. Sequence determination of the C. glutamicum gene for this enzyme revealed an open reading frame coding for a polypeptide of 436 amino acids. From this information and the molecular weight determination of the native enzyme, it follows that the dehydratase is a tetramer with a total mass of 186,396 daltons. Comparison of the deduced polypeptide sequence with the sequences of known threonine dehydratases revealed surprising differences from the C. glutamicum enzyme in the carboxy-terminal portion. This portion is greatly reduced in size, and a large gap of 95 amino acids must be introduced to achieve homology. Therefore, the C. glutamicum enzyme must be considered a small variant of threonine dehydratase that is typically controlled by isoleucine and valine but has an altered structure reflecting a topological difference in the portion of the protein most likely to be important for allosteric regulation.


Cloning and characterization of the Bacillus subtilis hemEHY gene cluster, which encodes protoheme IX biosynthetic enzymes.

Mutations that cause a block in a late step of the protoheme IX biosynthetic pathway, i.e., in a step after uroporphyrinogen III, map at 94 degrees on the Bacillus subtilis chromosomal genetic map. We have cloned and sequenced the hem genes at this location. The sequenced region contains six open reading frames: ponA, hemE, hemH, hemY, ORFA, and ORFB. The ponA gene product shows over 30% sequence identity to penicillin-binding proteins 1A of Escherichia coli, Streptococcus pneumoniae, and Streptococcus oralis and probably has a role in cell wall metabolism. The hemE gene was identified from amino acid sequence comparisons as encoding uroporphyrinogen III decarboxylase. The hemH gene was identified by enzyme activity analysis of the HemH protein expressed in E. coli. It encodes a water-soluble ferrochelatase which catalyzes the final step in protoheme IX synthesis, the insertion of ferrous iron into protoporphyrin IX. The function of the hemY gene product was not elucidated, but mutation analysis shows that it is required for a late step in protoheme IX synthesis. The hemY gene probably encodes an enzyme with coproporphyrinogen III oxidase or protoporphyrinogen IX oxidase activity or both of these activities. Inactivation of the ORFA and ORFB genes did not block protoheme IX synthesis. Preliminary evidence for a hemEHY mRNA was obtained, and a promoter region located in front of hemE was identified. From these combined results we conclude that the hemEHY gene cluster encodes enzymes for the synthesis of protoheme IX from uroporphyrinogen III and probably constitutes an operon.


CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus.

The czcR gene, one of the two control genes responsible for induction of resistance to Co2+, Zn2+, and Cd2+ (czc system) in the Alcaligenes eutrophus plasmid pMOL30, was cloned and characterized. The 1,376-bp sequence upstream of the czcCBAD structural genes encodes a 41.4-kDa protein, the czcR gene product, transcribed in the opposite direction of that of the czcCBAD genes. The putative CzcR polypeptide (355 amino acid residues) contains 11 cysteine and 14 histidine residues which might form metal cation-binding sites. A czcC::lacZ reporter gene translational fusion was constructed, inserted into plasmid pMOL30 in A. eutrophus, and expressed under the control of CzcR. Zn2+, Co2+, and Cd2+, as well as Ni2+, Cu2+, Hg2+, and Mn2+ and even Al3+, served as inducers of beta-galactosidase activity. Besides the CzcR protein, the membrane-bound CzcD protein was essential for induction of czc. The CzcR and CzcD proteins display no sequence similarity to two-component regulatory systems of a sensor and a response activator type; however, CzcD has 34% identity with the ZRC-1 protein, which mediates zinc resistance in Saccharomyces cerevisiae (A. Kamizomo, M. Nishizawa, Y. Teranishi, K. Murata, and A. Kimura, Mol. Gen. Genet. 219:161-167, 1989).


Menaquinone (vitamin K2) biosynthesis: evidence that the Escherichia coli menD gene encodes both 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase and alpha-ketoglutarate decarboxylase activities.

The formation of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC), the first identified intermediate in the menaquinone biosynthetic pathway, requires two reactions. They are the decarboxylation of alpha-ketoglutarate by an alpha-ketoglutarate decarboxylase, which results in the formation of succinic semialdehyde-thiamine PPi (TPP) anion, and the addition of the succinic semialdehyde-TPP anion to isochorismate carried out by the enzyme SHCHC synthase. Evidence is provided to support the conclusion that both enzymatic activities are encoded by an extended menD gene which is capable of generating a bifunctional 69-kDa protein. Consistent with the requirement for TPP in the decarboxylation of alpha-ketoglutarate, the translated amino acid sequence contains the characteristic TPP-binding motif present in all well-characterized TPP-requiring enzymes.


Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2.

The stable inheritance of the broad-host-range plasmid RK2 is due at least in part to functions within a region located at coordinates 32.8 to 35.9 kb, termed the RK2 par locus. This locus encodes four previously identified genes in two operons (parCBA and parD; M. Gerlitz, O. Hrabak, and H. Schwab, J. Bacteriol. 172:6194-6203, 1990, and R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). The parCBA operon is functional in resolving plasmid multimers to monomers. Analysis of the plasmid stabilization capacity of deletions within this region, however, indicates that this multimer resolution operon is required for stabilization only in certain Escherichia coli strains and under specific growth conditions. The deletion analysis further allowed a redefinition of the minimal functional region as 790 bp in length, consisting of the parD gene (243 bp) and its promoter as well as sequences downstream of parD. This minimal region stabilizes an RK2-derived minireplicon in several different gram-negative bacteria and, at least in E. coli, in a vector-independent manner. By insertional mutagenesis, both the parD gene and downstream (3') regions were found to be required for plasmid stabilization. The downstream DNA sequence contained an open reading frame which was subsequently shown by transcriptional and translational fusions to encode a protein with a predicted size of 11,698 Da, designated ParE. Since the parDE operon requires the presence of the parCBA operon for efficient stabilization under certain growth conditions, the potential role of multimer resolution in plasmid stabilization was tested by substituting the ColE1 cer site for the parCBA operon. While the cer site did function to resolve plasmid multimers, it was not sufficient to restore stabilization activity to the parDE operon under growth conditions that require the parCBA operon for plasmid stability. This suggests that plasmid stabilization by the RK2 par locus relies on a complex mechanism, representing a multifaceted stabilization system of which multimer resolution is a conditionally dispensable component, and that the function(s) encoded by the parDE operon is essential.


Three clustered origins of replication in a promiscuous-plasmid replicon and their differential use in a PolA+ strain and a delta PolA strain of Escherichia coli K-12.

A 1,197-bp region of the broad-host-range plasmid pCU1 is adequate for its replication. Analysis of replicating molecules containing this region reveals three clustered origins of vegetative replication and replication proceeds bidirectionally from each in a theta mode. In an Escherichia coli polymerase I deletion mutant, utilization of one of these three origins was not detected. The potentiality for origin utilization may therefore be a determinant of replicon host range.


The carboxy-terminal 14 amino acids of phage lambda N protein are dispensable for transcription antitermination.

The analogous N proteins encoded by lambdoid bacteriophages lambda, 21, and 22 are very different in amino acid sequence, except at their carboxy-terminal ends. Since N lambda remains functional despite the deletion of most of its terminal region of homology to N21, that region of homology cannot represent a region of conserved function.


Properties of purified sporlets produced by spoII mutants of Bacillus subtilis.

A number of abortively disporic spoII mutants of Bacillus subtilis released their forespore compartments (termed stage II sporlets) after mother cell lysis during sporulation in nutrient exhaustion or resuspension media. Stage II sporlets were viable and contained levels of ATP and a number of enzymes similar to those in cells 2 to 3 h after sporulation. However, stage II sporlets carried out essentially no macromolecular synthesis, a result suggesting that they were in a quiescent state. The nucleoid of these quiescent stage II sporlets was significantly condensed relative to that in the original vegetative cells, as was previously found to take place 1 to 2 h after initiation of sporulation (B. Setlow, N. Magill, P. Febbroriello, L. Nakhimousky, D. E. Koppel, and P. Setlow, J. Bacteriol. 173:6270-6278, 1991). Stage II sporlets may be a useful model system for analysis of forespore properties early in stage II of sporulation.


Rate maintenance of cell division in Escherichia coli B/r: analysis of a simple nutritional shift-down.

A competitive (nonmetabolizable) inhibitor of glucose uptake, alpha-methylglucoside, was used to limit the growth of Escherichia coli. Cell division during such a nutritional shift-down was studied in batch cultures and with the "baby-machine" technique. Following a brief delay, the rate of division was maintained for 60 to 70 min in batch cultures and for an extended period in the baby machine. Decreases in cell size were due, in part, to a possible reduction in the mass per chromosome origin at the time of replication initiation and a shorter time interval between initiation and the subsequent division. These unusual findings suggest that this method for abrupt change in growth rate without modifying repression patterns is useful for studying the control of various aspects of the bacterial cell.


Presence of methylated adenine in GATC sequences in chromosomal DNAs from Campylobacter species.

We digested chromosomal DNAs from 12 Campylobacter strains (C. jejuni, 4 strains; C. coli, 2 strains; C. fetus subsp. fetus, 2 strains; C. hyointestinalis, 2 strains; and C. upsaliensis, 2 strains) and from 4 Helicobacter strains (H. pylori, 2 strains; and H. mustelae, 2 strains) with HindIII, SstI, BamHI, DpnI, MboI, and Sau3AI. Restriction fragments were then separated by electrophoresis in 1% agarose or 10% polyacrylamide gels. Only DNAs from three Campylobacter species (C. jejuni, C. coli, and C. upsaliensis) were digested with DpnI (an enzyme that recognizes only methylated adenine in GATC sequences). We used MboI and Sau3AI to confirm these findings.


Structural and functional properties of the p60 proteins from different Listeria species.

The major extracellular protein p60 of Listeria monocytogenes seems to be required for this microorganism's adherence to and invasion of 3T6 mouse fibroblasts but not for adherence to human epithelial Caco-2 cells. Western blot analysis with polyclonal antibodies against p60 of L. monocytogenes indicated the presence of cross-reacting proteins in the culture supernatants of all Listeria species. Protein p60 of L. monocytogenes could restore adhesion of the L. monocytogenes mutant RIII (impaired in the synthesis of p60) to mouse fibroblasts more efficiently than that of Listeria grayi. The amino acid sequences of the p60-related proteins of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi indicated highly conserved regions of about 120 amino acids at both the N-terminal and the C-terminal ends. The middle portions of these proteins, consisting of about 240 amino acids, varied considerably. These parts include the repeat domain consisting of repetitions of Thr (T) and Asn (N) which was present only, albeit in different arrangements, in the p60 proteins of L. monocytogenes and L. innocua. The p60-related proteins of L. grayi, L. ivanovii, L. seeligeri, and L. welshimeri each contained an insertion of 54 amino acids which was absent in the p60 proteins of L. monocytogenes and L. innocua.


Evidence that the hemolysin/bacteriocin phenotype of Enterococcus faecalis subsp. zymogenes can be determined by plasmids in different incompatibility groups as well as by the chromosome.

The hemolysin (Hly/Bac) determinant in strains of Enterococcus faecalis was found to be present on plasmids in different incompatibility groups (conferring different sex pheromone responses) as well as on the chromosome. Of 33 Hly/Bac plasmids identified in clinical isolates, the related pheromone for 30 was cAD1; the related pheromone for another two (pYI1 and pYI3) or one (pYI2) was cOB1 or cY12, respectively. The representative Hly/Bac plasmids pAD1, pYI1, pOB1, and pYI2, which responded to pheromones cAD1, cOB1, cOB1, and cYI2, respectively, were compatible with one another. As additions to the incompatibility group IncHly of pAD1, groups for pOB1, pYI1, and pYI2 were designated IncHlyII, IncHlyIII, and IncHlyIV, respectively. Eleven of the 30 plasmids conferring a response to cAD1 were very similar to pAD1 on the basis of their restriction endonuclease profiles. EcoRI fragment D, F, or H containing parts of the Hly/Bac gene(s) of pAD1 hybridized to similar EcoRI fragments from each of the other three representatives of incompatibility groups (i.e., pOB1, pYI1, and pYI2) and to homologous DNA representing the chromosome of the plasmid-free Hly/Bac strain YI6-1.


Treatment outcome of obsessive compulsive disorder with comorbid social phobia.

Treatment of obsessive compulsive disorder (OCD) with serotonin reuptake blockers has been demonstrated effective in 50% to 60% of patients in open and placebo-controlled studies. However, some reports indicate that comorbid Axis II psychopathology, including avoidant personality disorder, and deficiency of social skills could be predictors of a poor response to treatment in OCD patients. A retrospective review elicited 12 patients who met DSM-III-R diagnostic criteria for both OCD and social phobia and were treated in our clinic last year with adequate trials of serotonin reuptake blockers or MAOIs. Only 3 (27%) of the 11 patients treated with serotonin reuptake blockers had a substantial improvement of OCD symptoms. Among them, only 1 (11%) of 9 patients with generalized subtype of social phobia versus 2 (100%) of 2 patients with the nongeneralized subtype responded to serotonin reuptake blockers. Four (80%) of 5 patients with comorbid generalized social phobia receiving phenelzine had marked improvement of OCD symptoms. In general, response of social phobia occurred parallel to that of OCD. Comorbid generalized social phobia seems to be associated with a poor response to serotonin reuptake blockers in OCD patients. Deficient social skills, as well as distinct biological mechanisms, may be involved. MAOIs might be an effective alternative medication in refractory cases. Larger and controlled studies are needed to define the implications of the association of OCD and social phobia.


Night terrors in adults: phenomenology and relationship to psychopathology.

Night terrors have been classically described in children. Night terrors occurring in adults have been linked to psychopathology. Recent descriptions of sleep panic attacks have raised questions about their relationship to night terrors. Evaluations from a medically affiliated sleep disorders program were reviewed to identify adult patients presenting with events consistent with night terrors. Eleven patients were identified, 10 of whom had polysomnographic evaluation, and their records were reviewed for information relevant to night terrors and psychiatric symptoms. Six of these patients were available for further assessment which included inquiry regarding sleep events, a Structured Clinical Interview (SCID) for psychiatric disorders, and the Millon Clinical Multiaxial Inventory II (MCMI-II) for personality-related measurements. In the original sample, night terror episodes featured confused behaviors, motor activity, and absent or fragmented recall. Polysomnography documented arousals from slow wave sleep in 9 of 10 patients. All of the original patients reported psychiatric symptoms. All 6 patients who received the subsequent structured evaluation met lifetime criteria for Axis I conditions (most commonly affective and substance use disorders) and had elevated scores on the personality scales of the MCMI-II. Night terrors were not limited to psychiatric episodes. Night terrors occur in adults that are similar to episodes described in children. While distinct from sleep panic attacks, night terrors appear to occur in adults with histories of psychopathology.


Restraint, seclusion, and clozapine.

The effects of clozapine, an unconventional neuroleptic drug, on the use of restraint and seclusion in patients have not been extensively reported on in the United States. The records of 107 patients receiving clozapine in Missouri state mental hospitals were reviewed over a 13-month period for frequency and duration of restraint and seclusion. During clozapine treatment, patients had fewer episodes of restraint and seclusion than previously. The duration of restraints and seclusions also decreased, starting the second month after the initiation of clozapine treatment. For patients who were restrained or secluded before clozapine treatment, the decrease in the number and duration of restraint and seclusion episodes was dramatic.


The effect of enalapril on serum lithium levels in healthy men.

Several authors have recently described the development of lithium toxicity after the addition of angiotensin-converting enzyme inhibitors to a stable lithium regimen. This pilot study was designed to systematically investigate the effects of enalapril, an angiotensin-converting enzyme inhibitor, on the serum level of lithium. In a 26-day outpatient study, nine healthy men took lithium for 10 days, lithium and enalapril for 10 days, and lithium alone again for 6 days. Serum lithium levels were measured while patients were taking lithium alone and while they were taking the lithium/enalapril combination. There were no statistically significant differences between mean serum lithium level during treatment with lithium alone and during treatment with the lithium/enalapril combination. However, one subject showed a 31% increase in serum lithium level after enalapril was added. Although no statistically significant differences between mean serum lithium level during treatment either with lithium alone or with lithium/enalapril were found, it is possible that the low dose of enalapril and low serum lithium levels employed for subject safety may have resulted in a type II statistical error. If enalapril doses or initial serum lithium levels were similar to those described in case reports, a significant difference in mean serum lithium levels may have been observed. While a predictable interaction between lithium and enalapril probably does not occur in all patients, factors such as enalapril dose, serum lithium level before addition of enalapril, or the presence of heart disease may make such an interaction more likely. At the levels of lithium and enalapril used in this study, elevated serum lithium concentration does not appear to be a universal event, but physicians must exercise appropriate caution.


Personality and clinical characteristics in patients with trichotillomania.

Trichotillomania, a disorder of self-directed hair pulling, has been the subject of few systematic studies. Although personality characteristics and disorders are often noted to coexist with trichotillomania, no thorough assessment of comorbidity with DSM-III-R Axis II disorders has been published. The present study was conducted to evaluate personality disorders and other personality characteristics in a large outpatient population of trichotillomanics and to compare these findings with those in a nontrichotillomanic comparison group. Forty-eight outpatient female trichotillomanics were evaluated with the Structured Interview for DSM-III-R Personality Disorders (SIDP-R) and the revised version of the Minnesota Multiphasic Personality Inventory (MMPI-2). Personality disorders and personality cluster symptom scores from the SIDP-R and MMPI-2 scales were compared with those derived from a comparison group of 48 age-matched female outpatient psychiatric patients. Forty-two percent of the trichotillomanic group met criteria for a personality disorder. The only statistically significant difference in frequency of diagnoses between the trichotillomanic and comparison groups was a greater frequency of borderline personality disorder in the comparison group. Trichotillomanics demonstrated significantly less SIDP-R cluster A personality symptoms as well as less depression and better psychological adjustment on the MMPI-2. Our study suggests that no particular personality disorder or trait characterizes female trichotillomanics. Female trichotillomanics seeking psychiatric intervention appear to have better psychological adjustment and less psychopathology in general than other psychiatric outpatients.


Comparison of SPECT applications in neurology and psychiatry.

The following presentation provides an overview of SPECT brain imaging applications in both neurologic and psychiatric disorders. Specific findings in cerebrovascular disease, dementia, and epilepsy are described in order to highlight the role SPECT plays in distinguishing primary organic disease from primary psychopathology. Through case examples, new applications of three-dimensional SPECT rCBF imaging that could play a major role in defining CNS network abnormalities in major depressive disorder and obsessive compulsive disorder are elucidated. The role of network imaging analysis across groups in psychiatric subjects is contrasted to its roles in neurologic disorders. The degree to which both neurologic and psychiatric pathology seen on SPECT exceeds that observed through structural modalities is defined.


Regional cerebral blood flow imaging with SPECT in psychiatric disease: focus on schizophrenia, anxiety disorders, and substance abuse.

In psychiatric disorders where gross brain structure is preserved, as confirmed through computed tomography and magnetic resonance imaging, neuroSPECT imaging provides further diagnostic insight by demonstrating abnormal brain function related to disturbances in perfusion or metabolism. This paper will focus on how the psychiatrist may incorporate SPECT brain imaging into the diagnostic process for schizophrenia, where frontal lobe hypoperfusion and hypometabolism have been demonstrated; for obsessive compulsive disorder, which has been linked to abnormalities in the basal ganglia, anterior cingulate gyrus, and orbital frontal cortex; and for substance abuse, where use of cocaine can be associated with cortical and subcortical perfusion defects.


Current and future applications of SPECT in clinical psychiatry.

Over the past 10 years several studies have been conducted in psychiatric and neurologic patients with single photon emission computed tomography (SPECT) to determine if patterns of brain dysfunction exist that characterize the different mental diseases. Although there has not been any finding that can be referred to as specific for a particular disease, SPECT studies have been able to demonstrate evidence of brain dysfunction in patients who, when tested with other means, showed no evidence of brain abnormalities. In this manuscript, the current and future applications of SPECT in the clinical practice of psychiatry are analyzed.


Neuroreceptor imaging with SPECT.

Single photon emission computed tomography (SPECT) imaging can provide useful measurements of brain receptors and endogenous neurotransmitters and may have significant experimental and clinical applications. This presentation reviews the use of SPECT for neuroreceptor imaging. Studies of receptors for benzodiazepines, dopamine D2 agents, and dopamine reuptake sites will be used to exemplify the capabilities of SPECT. Tracers labeled with the radioisotope 125I have high affinity, high brain uptake, and high ratios of specific to nonspecific binding. Imaging studies of human and nonhuman primate brain will be presented, and the potential clinical applicability of these agents will be discussed.


An introduction and overview to clinical applications of neuroSPECT in psychiatry.

This brief paper introduces the reader to the materials presented in other manuscripts of this issue of The Journal of Clinical Psychiatry. Single photon emission computed tomography (SPECT) is a dynamic brain imaging technique that grew out of the evolution of increasingly more sophisticated approaches to understanding the pathophysiology and biology of neurologic and psychiatric disorders over the past two decades. SPECT is a practical tool that is more available than positron emission tomography (PET), which is found primarily at academic centers. While SPECT has drawbacks that relate to levels of resolution and the possible impact of altered brain physiology on interpretation of results, it offers advantages of decreased cost, the need for less intensively trained technicians, and the ability to carry out clinical testing in facilities lacking a cyclotron. These factors raise the hope of more routine use of this imaging technique in clinical settings related to cognitive disorders, schizophrenia, affective disorders, and problems associated with substance use.


Brain SPECT imaging and psychiatry.

To provide a basis for using neuroSPECT (single photon emission computerized tomography) in the evaluation of psychiatric disorders, this presentation will focus on recent developments in this field. Newer advances in cerebral SPECT instrumentation and radiopharmaceuticals and the potential impact on the evaluation of psychiatric patients will be discussed.


Energetics of methanogenesis studied in vesicular systems.

Methanogenesis is restricted to a group of prokaryotic microorganisms which thrive in strictly anaerobic habitats where they play an indispensable role in the anaerobic food chain. Methanogenic bacteria possess a number of unique cofactors and coenzymes that play an important role in their specialized metabolism. Methanogenesis from a number of simple substrates such as H2 + CO2, formate, methanol, methylamines, and acetate is associated with the generation of transmembrane electrochemical gradients of protons and sodium ions which serve as driving force for a number of processes such as the synthesis of ATP via an ATP synthase, reverse electron transfer, and solute uptake. Several unique reactions of the methanogenic pathways have been identified that are involved in energy transduction. Their role and importance for the methanogenic metabolism are described.


Photophosphorylation elements in halobacteria: an A-type ATP synthase and bacterial rhodopsins.

Photophosphorylation in halobacteria is carried out by two rather simple elements: an A-type ATP synthase and light-driven ion-pumping bacterial rhodopsins. The unique features of halobacterial ATP synthase, mostly common to archaebacteria (A-type), and of new members of the bacteriorhodopsin family are introduced along with studies performed in the authors' laboratory. This is the story of how we found that the A-type ATP synthase is close to V-type ATPase but far from F-type ATPase, although all three ATPases are believed to have the same ancestor. Archaerhodopsins, the new members of the proton-pumping retinal proteins, were found in Australian halobacteria and have been used in a comparative study of bacterial rhodopsins.


Structures of archaebacterial membrane lipids.

Structural data on archaebacterial lipids is presented with emphasis on the ether lipids of the methanogens. These ether lipids normally account for 80-95% of the membrane lipids with the remaining 5-20% of neutral squalenes and other isoprenoids. Genus-specific combinations of various lipid core structures found in methanogens include diether-tetraether, dietherhydroxydiether, or diether-macrocyclic diether-tetraether lipid moieties. Some species have only the standard diether core lipid, but none are known with predominantly tetraether lipids as found in certain sulfur-dependent archaebacteria. The relative proportions of these lipid cores are known to vary in relation to growth conditions in Methanococcus jannaschii and Methanobacterium thermoautotrophicum. Polar headgroups in glycosidic or phosphodiester linkage to the sn-1 or sn-1' carbons of glycerol consist of polyols, carbohydrates, and amino compounds. The available structural data indicate a close similarity among the polar lipids synthesized within the species of the same genus. Detection of lipid molecular ions by mass spectrometry of total polar lipid extracts is a promising technique to provide valuable comparative data. Since these lipid structures are stable within the extreme environments that many archaebacteria inhabit, there may be specific applications for their use in biotechnology.


Structural features of archaebacterial cell envelopes.

Regularly arrayed surface (glyco) proteins--often referred to as S layers--are a common feature of the cell envelopes of almost all archaebacteria. We have selected some examples (Halobacterium, Sulfolobus, Thermoproteus, Pyrobaculum, Staphylothermus), and we describe the structure of their surface layers as revealed primarily by electron crystallography. In spite of a considerable diversity in shapes and dimensions, some common structural features emerge from the comparison. The glycoprotein arrays are composed of oligomeric units which are anchored in the plasma membrane; extended spacer or linker domains maintain the bulk of the more or less porous surface layers at a constant distance above the membrane surface, thus creating a quasi-periplasmic compartment. Functions ascribed to surface layers, such as compartmentalization, shape maintenance and determination, and adhesion are discussed.


Function and biosynthesis of gas vesicles in halophilic Archaea.

The proteinaceous gas vesicles produced by various microorganisms including halophilic Archaea are hollow, gas-filled structures with a hydrophobic inner and a hydrophilic outer surface. The structural components of gas vesicles and their biosynthesis are still under investigation; an 8-kDa polypeptide appears to be the major constituent of the gas-vesicle envelope. Genetic analysis of the halobacterial gas-vesicle synthesis revealed an unexpected complexity: about 14 genes organized in three transcription units are involved in gas-vesicle structure, assembly, and gene regulation. Here we describe the comparison of three different genomic regions encoding gas vesicles in Halobacterium salinarium (p-vac and c-vac regions) and Haloferax mediterranei (mc-vac region) and speculate on the function of the gene products involved in gas-vesicle synthesis.


Energy transduction and transport processes in thermophilic bacteria.

Bacterial growth at the extremes of temperature has remained a fascinating aspect in the study of membrane function and structure. The stability of the integral membrane proteins of thermophiles make them particularly amenable to study. Respiratory enzymes of thermophiles appear to be functionally similar to the mesophilic enzymes but differ in their thermostability and unusual high turnover rates. Energy coupling at extreme temperatures seems inefficient as suggested by the high maintenance coefficients and the high permeability of the cell membrane to protons. Nevertheless, membranes maintain their structure at these extremes through changes in fatty acid acyl chain composition. Archaebacteria synthesize novel membrane-spanning lipids with unique physical characteristics. Thermophiles have adapted to life at extreme temperatures by using sodium ions rather than protons as coupling ions in solute transport. Genetic and biochemical studies of these systems now reveal fundamental principles of such adaptations. The recent development of reconstitution techniques using membrane-spanning lipids allows a rigorous biochemical characterization of membrane proteins of extreme thermophiles in their natural environment.


Correlation between active form and dimeric structure of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart.

The active form of purified mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated by crosslinking with dimethylsuberimidate and SDS-PAGE, with or without pretreatment with the inactivating detergent Triton X-100. In the absence of detergent, crosslinked isomers of the dimeric form of 208-235 kDa were obtained. Addition of detergent led to the simultaneous loss of the dimers and the bulk of the activity. Removal of the detergent led to a partial restoration of both activity and the dimeric forms. The results suggest that the active form is a dimer, and that the detergent-dependent conversion to the largely inactive monomer is reversible. It is proposed that the mechanism of inactivation of transhydrogenase by Triton X-100 involves a disruption of essential hydrophobic interactions between the membrane-spanning regions of the monomers.


Differential scanning calorimetry study of glycogen phosphorylase b-detergent interactions.

The overall thermal denaturation of glycogen phosphorylase b is irreversible and our results conform to the theoretical prediction of a reversible process followed by a slower irreversible process. The basic thermodynamic parameters of glycogen phosphorylase b denaturation have been worked out and found to be: critical temperature 57.0 +/- 0.5 degrees C, transition half-width 8 +/- 1 degrees C, and calorimetric enthalpy change and Van't Hoff enthalpy change of the denaturation process 450 +/- 50 and 105 +/- 15 kcal/mol of enzyme monomer, respectively, at pH 7.4. These parameters have been found to be largely altered by the detergents octylglucoside, cholate, and deoxycholate at or below their critical micelle concentration, but not by Triton X-100 nor by lecithin liposomes. Organic solvents, such as dimethyl sulfoxide and methanol, and the presence of sarcoplasmic reticulum membranes produces an alteration of the denaturation thermogram of glycogen phosphorylase b similar to that produced by the above-mentioned detergents. These results allow us to hypothesize that hydrophobic domains of glycogen phosphorylase b are involved in its association to sarcoplasmic reticulum membranes in the sarcoplasmic reticulum/glycogenolytic complex of mammalian skeletal muscle.


Characterization of the inhibitor sensitivity of the coenzyme A transport system in isolated rat heart mitochondria.

The effect of protein labeling agents on coenzyme A (CoA) transport into isolated rat heart mitochondria was studied. CoA transport was substantially inhibited by sulfhydryl reagents (mersalyl, pCMB) as well as by the tyrosine-selective reagent N-acetylimidazole. The effect of pCMB was reversed by DTT. Moreover, CoA uptake was completely abolished by agents selective for lysine and amino terminal residues (pyridoxal 5-phosphate, dansyl chloride). In contrast arginine-selective reagents (2, 3-butanedione, phenylglyoxal) caused considerably less inhibition of CoA uptake. Moreover, partial inhibition of transport was observed with the stilbene disulfonic acid derivatives DIDS and SITS. Finally, measurement of the effects of the labeling agents on the mitochondrial membrane potential indicated that the inhibition of CoA transport into mitochondria is not a secondary effect that arises from an alteration in the electric potential gradient across the inner mitochondrial membrane. These results provide the first information on the types of amino acid residues that may be essential to the CoA transport mechanism and provide additional support for the existence of a CoA transport protein within the mitochondrial inner membrane. Furthermore, the identification of effective inhibitors of the CoA transport system will greatly facilitate the functional reconstitution of this transporter in a proteoliposomal system following its solubilization and purification.


Negative regulation of hepatocyte growth factor gene expression in human lung fibroblasts and leukemic cells by transforming growth factor-beta 1 and glucocorticoids.

Hepatocyte growth factor (HGF), a mesenchymal-derived factor which regulates growth, motility, and morphogenesis of epithelial and endothelial cells, functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. We have now obtained evidence that transforming growth factor-beta 1 (TGF-beta 1) and glucocorticoids are negative regulators for HGF gene expression. When TGF-beta 1 or dexamethasone was added to cultures of MRC-5 human embryonic lung fibroblasts and HL-60 human promyelocytic leukemic cells, the amount of HGF secreted into the culture medium was inhibited to 30-40% of that of control cultures by 10 ng/ml TGF-beta 1 and to 40-50% by 10(-6) M dexamethasone. The inhibitory effect of TGF-beta 1 and dexamethasone on HGF synthesis in MRC-5 cells was additive, thereby suggesting that TGF-beta 1 and dexamethasone exert effects through distinct mechanisms. Hydrocortisone also inhibited HGF synthesis with the same potency as dexamethasone; however, testosterone, estriol, and beta-estradiol had no effect. The rate of HGF synthesis in MRC-5 cells, as measured by pulse labeling with [35S]methionine and subsequent immunoprecipitation, was suppressed to 30-40% of the control with 10 ng/ml TGF-beta 1, and to 30-45% by 10(-6) M dexamethasone. HGF mRNA levels in MRC-5 cells and HL-60 cells were dose-dependently suppressed by TGF-beta 1 and dexamethasone; 10 ng/ml TGF-beta 1 suppressed HGF mRNA levels to 32% and 35% of control culture, respectively, in MRC-5 cells and HL-60 cells, and 10(-6) M dexamethasone suppressed to 43% and 38%, respectively. Thus, TGF-beta 1 and glucocorticoids seem to inhibit HGF synthesis by suppressing the expression of the HGF gene. We propose that a negative regulation of HGF gene expression by TGF-beta 1 or glucocorticoids may be involved in physiological or pathological processes during tissue regeneration.


Thrombospondin 1 and thrombospondin 2 are expressed as both homo- and heterotrimers.

There exist two distinct thrombospondin molecules (designated TSP1 and TSP2) which are encoded by separate genes. TSP1 is a trimeric cell surface and extracellular matrix molecule. Sequence comparison reveals that the 2 cysteines involved in interchain disulfide linkage and trimer assembly in TSP1 are conserved in TSP2 (Laherty, C. D., O'Rourke, K., Wolf, F. W., Katz, R., Seldin, M. F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 3274-3281). Swiss 3T3 fibroblasts express both TSP1 and TSP2, and, therefore, an important question is whether TSP in such cells is expressed as homotrimers or as heterotrimers. We find that Swiss 3T3 cells and epithelial cells transfected with TSP expression vectors express both homo- and heterotrimeric forms of TSP. In addition, homotrimeric TSP2 has a lower affinity for heparin than homotrimeric TSP1. Thus, the heparin affinity of TSP can be modulated by the expression of TSP as homo- or heterotrimers.


Inhibition of glyoxalase I by the enediol mimic S-(N-hydroxy-N-methylcarbamoyl)glutathione. The possible basis of a tumor-selective anticancer strategy.

In principle, competitive inhibitors of glyoxalase I that also serve as substrates for the thioester hydrolase glyoxalase II might function as tumor-selective anti-cancer agents, given the role of these enzymes in removing cytotoxic methylglyoxal from cells and the observation that glyoxalase II activity is abnormally low in some types of cancer cells. In support of the feasibility of this anticancer strategy, an inhibitor of this type has been synthesized by a thioester-interchange reaction between glutathione and N-hydroxy-N-methylcarbamate 4-chlorophenyl ester to give S-(N-hydroxy-N-methylcarbamoyl)glutathione (1). This compound was designed to be a tight-binding inhibitor of glyoxalase I, on the basis of its stereoelectronic similarity to the enediol(ate) intermediate that forms along the reaction pathway of this enzyme. Indeed, 1 is a competitive inhibitor of yeast glyoxalase I, with an inhibition constant (Ki = 68 microM) that is approximately 30-fold lower than that reported for S-D-lactoylglutathione and approximately 7-fold lower than the Km for glutathione-methylglyoxal thiohemiacetal. In addition, 1 is a substrate for bovine liver glyoxalase II, with a Km (0.48 mM) approximately equal to that of the normal substrate S-D-lactoyglutathione and a kcat approximately 2 x 10(-5)-fold that of the normal substrate. Membrane transport studies show that 1 can be delivered into human erythrocytes (used here as a model cell) either by direct diffusion of 1 across the cell membrane or by more rapid diffusion of the glycylethyl ester of 1 across the cell membrane, followed by the catalyzed hydrolysis of the ester to give 1.


Determinants for DNA-binding site recognition by the glucocorticoid receptor.

The glucocorticoid receptor binds with high specificity to glucocorticoid response elements, discriminating them from other closely related binding sites. Three amino acids in the recognition alpha-helix of the DNA-binding domain of the receptor are primarily responsible for this specific DNA binding activity. In this study we analyze in detail how these residues determine the specific DNA binding by studying a series of mutant glucocorticoid receptor DNA-binding domains containing all combinations of glucocorticoid and estrogen receptor-specific residues at these positions. Statistical analysis of the results enables us to create models describing the association between amino acids and base pairs. Several strategies appear to be used in accomplishing discrimination between the glucocorticoid and estrogen response elements. Single residues (i.e., Val-443 in the glucocorticoid receptor and Glu-439 in the estrogen receptor) appear to form both positive contacts with specific base pairs in the cognate binding site and negative contacts in the non-cognate site. In the glucocorticoid receptor Ser-440 is pleiotropically negative for all sites tested but the negative effect is stronger for the estrogen response element thus contributing to binding site discrimination. Furthermore, combinations of amino acids appear to act synergistically, most often causing a reduction in binding to non-cognate sites.


Characterization and purification of a novel transcriptional repressor from HeLa cell nuclear extracts recognizing the negative regulatory element region of human immunodeficiency virus-1 long terminal repeat.

Cellular transcription factors play critical roles in regulating human immunodeficiency virus (HIV) gene transcription, although the precise mechanism(s) defining their roles are not well established. Primarily it has been suggested that sequence-specific interaction of trans-activating proteins with cis-acting DNA elements plays a crucial role in regulating the target genes. The negative regulatory element (NRE) of HIV-1 long terminal repeat (LTR) is one such defined region that has been reported to down-regulate LTR-directed HIV gene expression. Information regarding the role of this region in the regulation of HIV expression is lacking. Here we describe an attempt to further characterize the role of NRE cis-elements and define any sequence-specific interaction with cellular factors. Using gel mobility shift DNA-binding and Southwestern blot assays, we have mapped a distinct region of NRE (-290 to -260, a 30-base pair (bp) domain of NRE-A) sequences of HIV-1 LTR, which recognizes a specific DNA-binding protein from HeLa cell nuclear extracts. This factor is a 38-kDa polypeptide which can be affinity-purified to near homogeneity by this 30-bp specific oligonucleotide in affinity chromatography. The cellular factor from HeLa cell nuclear extract exhibits specific interaction only with the 30-bp NRE-A domain of HIV-1 LTR and acts as a strong transcriptional repressor/inhibitor molecule in the DNA-protein gel binding, as well as in vitro transcriptional studies with the nuclear extracts from cells with productive HIV-1 infection. To our knowledge, this is the first report of a factor recognizing a distinct segment within NRE that has been shown to exert an inhibitory effect on transcriptionally active DNA-protein "pre-initiation" complex formation, suggesting a possible role in HIV-1 gene regulation.


Ultraviolet-induced dimerization of non-adjacent pyrimidines in poly[d(A-T)].

The DNA photoproduct responsible for the ultraviolet (UV) light-induced -1 frameshift mutation remains unknown. We recently identified a UV photoproduct consisting of a cyclobutane dimer occurring between non-adjacent thymine residues in the same strand, [sequence: see text] and proposed that replication across this unrepaired photoproduct might result in a -1 frameshift mutation since the intervening base is extrahelical. Until now this novel photoproduct has only been identified in single-stranded DNA polymers and does not occur in UV-irradiated double-stranded polymers due to conformational restraint. This observation suggested that this photoproduct could only occur in vivo in chromosomal sites that were single-stranded. In the current work the cis-syn dithymine cyclobutane dimer has been identified in the self-complementary polymer poly[d(A-T)] when UV irradiated in solution conditions (concentrated manganese chloride or 60% ethanol plus trace salts) wherein this polymer remains double-stranded but the double-helix is partially destabilized. Taken together, the current findings suggest that dipyrimidine photoproducts between non-adjacent residues on the same strand could occur in vivo in double-stranded, but partially destabilized, DNA.


Cytotoxicity of momordin-folate conjugates in cultured human cells.

We have shown previously that macromolecules can be nondestructively delivered into cultured cells via folate receptor-mediated endocytosis if the macromolecules are conjugated to folic acid prior to addition to receptor-bearing cells (Leamon, C.P., and Low, P. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5572-5576). Although an intracellular destination of the folate-linked proteins could be easily documented, the spatial resolution of the earlier data was insufficient to evaluate whether any endocytosed material was delivered into the cytosol. To resolve this issue, a folate-toxin conjugate was constructed using the impermeable ribosome-inactivating protein, momordin. Diminution of [3H]leucine incorporation into newly synthesized protein was then employed as a quantitative measure of the entry of the toxin into the cytosol. In studies with both HeLa and KB cells, cellular protein synthesis was found to be inhibited in a time- and concentration-dependent manner by the momordin-folate conjugate, but not by the underivatized toxin. IC50 values centered around 10(-9) M for the folate-linked samples. These observations provide direct evidence that folate conjugates not only reach the cytosol, but do so in a functionally active form.


Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning.

Cloning of the sorbitol dehydrogenase gene (gutB) from Bacillus subtilis offers an excellent system for studying zinc binding, substrate specificity, and catalytic mechanism of this enzyme through protein engineering. As a first step to clone gutB, B. subtilis sorbitol dehydrogenase has been purified to homogeneity and characterized. It is a tetrameric enzyme with a molecular mass of 38 kDa for each subunit. Atomic absorption analysis shows the presence of 1 mol of zinc atom/subunit. Substrate specificity and stereospecificity of the enzyme toward C-2 and C-4 of hexitols were established. Sequence of the first 31 amino acids was determined, and a set of oligonucleotide probes was designed for gene cloning. A positive clone carrying a 5-kilobase pair HindIII insert was isolated and sequenced. Sequence alignment indicated that the deduced amino acid sequence of B. subtilis sorbitol dehydrogenase shows 36% identity in sequence with the liver sorbitol dehydrogenase from sheep, rat, and human. In reference to the sequence of alcohol dehydrogenase, two potential zinc binding sites were identified. Sequence information related to the structure-function relationships of the enzyme is discussed.


The AMP-binding domain on adenylate kinase. Evidence for a conformational change during binary-to-ternary complex formation via photoaffinity labeling analyses.

The topological location of the nucleotide substrate binding environments on adenylate kinase has been explored with the fluorescent molecule [4-benzoyl]benzoyl-1-amidofluorescein (BzAF) and the nucleotide analog 3'-O-[4-benzoyl]benzoyl-ATP (BzATP), which are site-directed photoaffinity probes that bind covalently at the individual nucleotide sites. The MgBzATP substituted for MgATP as a substrate for this enzyme, whereas BzAF, which is neither a substrate nor a nucleotide, behaved as a competitive inhibitor for each nucleotide site independently. BzAF could be directed specifically to either of the nucleotide sites by near saturation of the second site with its natural nucleotide substrate. Using this second site blocking approach, each nucleotide site, in turn, could be protected competitively from BzAF-induced photoinhibition by the presence of its natural substrate. This strategy showed that under photolysis, the Kd(BzAF) of 0.1 mM (for the MgATP site) and 0.2 mM (for the AMP site) were nearly identical with the Km(app) values determined for MgATP and AMP, respectively. Pseudo first-order photolysis kinetics with [3H]BzAF revealed covalent binding stoichiometries for full inhibition of 1 mol of probe/mol of enzyme at either site. Thus, we prepared the binary complex ([3H]BzAF-enzyme) by photo-labeling the MgATP site-blocked enzyme with [3H]BzAf to 1:1 molar stoichiometry. Tryptic digestion followed by partial sequencing of the [3H]BzAF-labeled enzyme disclosed that BzAF was bound specifically within the peptide span Gly64-->Arg77. This amino acid domain therefore probably constitutes the neighborhood identifiable with AMP binding. Furthermore, we also achieved double photocovalent labeling of adenylate kinase with both photoprobes, by first cross-linking with approximately 1 mol of MgBzATP, followed by approximately 0.9 mol of [3H]BzAF, thus generating a ternary covalent complex. Comparison of the fluorescence of the binary species and the ternary (BzATP-enzyme-[3H]BzAF) complex revealed altered fluorescence emission profiles, which could indicate that a conformational change occurs during formation of the ternary (or transition state) complex.


The influence of flanking sequence on the O-glycosylation of threonine in vitro.

To investigate the influence of flanking amino acid sequence on the O-glycosylation of a single threonine residue in vitro, we have examined a series of 52 related peptides. The substrates were based upon a sequence from human von Willebrand factor which is known to be glycosylated in vivo (-6PHMAQVTVGPGL+5). Each residue of the parent peptide was substituted, in turn, with isoleucine, alanine, proline, glutamic acid, or arginine. Peptides were glycosylated using a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase purified 15,000-fold from bovine colostrum by chromatography on DEAE-Sephacel, SP-Sephadex, Sephacryl S-300, Affi-Gel Blue, and 5-mercuri-UDP-GalNAc thiopropyl-Sepharose. Single amino acid changes in the sequences flanking the threonine could profoundly alter the glycosylation of the substrate peptides. Substitution of any amino acid tested at positions +3, -3, and -2 markedly decreased O-glycosylation, as did the presence of a charged residue at position -1. The substitution of amino acids at the other positions of the peptide substrate had little effect on the incorporation of GalNAc. Statistical analysis of sequences flanking known glycosylated threonine and serine residues suggests that they should be glycosylated with equal efficiency in the same sequence context (O'Connell et al., 1991). However, the bovine colostrum transferase failed to glycosylate a peptide derived from human erythropoietin which contains a serine that is glycosylated in vivo (-5PPDAASAAPLR+5). When a threonine was substituted for the serine in this peptide (-5PPDAATAAPLR+5), the substrate proved to be an excellent acceptor of GalNAc. These observations indicate that although flanking amino acid sequence is important for the O-glycosylation of specific hydroxyamino acids, discrete threonine- and serine-specific transferases may exist.


Characterization of a novel, potent, and specific inhibitor of serine palmitoyltransferase.

We have examined the mechanism of action of two natural products identified as broad spectrum antifungal agents (VanMiddlesworth, F., Dufresne, C., Wincott, F. E., Mosley, R. T., and Wilson, K. E. (1992) Tetrahedron Lett., in press; VanMiddlesworth, F., Giacobbe, R. A., Lopez, M. Garrity, G., Bland, J. A., Bartizal, K., Fromtling, R. A., Polishook, J., Zweerink, M. M., Edison, A. M., Rozdilsky, W., Wilson, K. E., and Monaghan, R. L. (1992) J. Antibiot. (Tokyo) 45, 861-867), designated sphingofungin B (2S-amino-3R,4R,5S,14-tetrahydroxyeicos-6-enoic acid) and sphingofungin C (2S-amino-5S-acetoxy-3R,4R,14-trihydroxyeicos-6-enoic acid), and find they are potent specific inhibitors of serine palmitoyltransferase, which catalyze the committed step of sphingolipid biosynthesis. We used Saccharomyces cerevisiae as a model to investigate the mechanism of the antifungal activity of these compounds. Macromolecular synthesis was not immediately affected by either sphingofungin B or C, synthesis continued for 60-90 min following the addition of drug to growing cultures. Significant loss of viability with sphingofungins required growing cultures and began only after several hours, with greater than 99.9% of drug-treated cells non-viable after 24 h. No lysis or other gross changes in cell morphology were observed in drug-treated cells. The structural similarity of sphingofungin B and C to sphingosine and phytosphingosine prompted us to investigate their effects on sphingolipid synthesis. Nanomolar levels of the drugs inhibited the incorporation of [3H]inositol into sphingolipid before incorporation into the sphingolipid precursor, phosphatidylinositol was affected, suggesting specific inhibition of sphingolipid synthesis. This hypothesis was confirmed by experiments in which the growth inhibitory activity of both drugs was completely ablated by the addition of phytosphingosine, dihydrosphingosine, or ketodihydrosphingosine to the culture medium. Reversal of antifungal activity by ketodihydrosphingosine suggested that serine palmitoyltransferase could be the actual target of these compounds. Direct evidence for this hypothesis was the observation of inhibition of serine palmitoyltransferase activity in crude membrane preparations at nanomolar concentrations of each drug. The potent inhibition of serine palmitoyltransferase coupled with the apparent lack of effect of these compounds on other cellular functions suggests that sphingofungin B and C will prove to be important new tools for studying the role of sphingolipids in yeast and perhaps in other organisms.


Inhibition of carbonic anhydrases I and II by N-unsubstituted carbamate esters.

We previously showed that the zinc metalloenzyme carbonic anhydrases (CA I and II isozymes) bind "neutral" amides and related compounds as anions through coordination of their deprotonated amide nitrogen to the active site zinc (Rogers, J. I., Mukherjee, J., and Khalifah, R. G. (1987) Biochemistry 26, 5672-5679). Urethan, the ethyl carbamate ester, was among such compounds. The present study was designed to test whether other N-unsubstituted carbamate esters of pharmacological interest (as sedatives, hypnotics, anxiolytics, and skeletal muscle relaxants) were capable of binding to CA in the same manner. We studied the interaction of human CA I and II with urethan, phenyl carbamate, ethinamate, meprobamate, and methocarbamol. Phenyl carbamate studies were greatly complicated by its uncatalyzed hydrolysis via an elimination mechanism to form cyanate, a powerful CA inhibitor. In general, the compounds display: 1) slow on-off inhibition binding kinetics in the seconds range, 2) maximal inhibitor affinity at alkaline pH, and 3) characteristic three-band visible spectra of their complexes with cobalt-substituted CA I. These properties are shared with the previously studied amide inhibitors and are taken as evidence that the deprotonated carbamate nitrogen coordinates to the active site metal ion. CA I appeared to bind carbamate esters more tightly than CA II, an unusual 1000-fold selectivity being seen in the case of methocarbamol. The inhibition by these drugs is not sufficiently strong to implicate CA I and II in their mechanism of action. However, it does suggest the possible existence of previously unsuspected similarities between binding to CA and to their physiological receptors or targets, particularly the involvement of zinc.


Structural and functional relationships in DnaK and DnaK756 heat-shock proteins from Escherichia coli.

The secondary structures of DnaK and the mutant DnaK756 heat-shock proteins from Escherichia coli have been investigated by Fourier transform infrared spectroscopy. The analysis of infrared data showed that DnaK and DnaK756 proteins have different secondary structures that are not affected by the presence of ATP or beta, gamma-methyleneadenosine 5'-triphosphate. The infrared data indicate also that the tertiary structures of DnaK and DnaK756 proteins are different and that DnaK protein undergoes conformational changes in its tertiary structure not only during binding of ATP but also during ATP hydrolysis. Using fluorescence spectroscopy of a single tryptophan located in the N-terminal domain of DnaK protein and fluorescence of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, which interacts with hydrophobic domains of DnaK protein, we were able to distinguish between two conformational states of DnaK protein. After binding of triphosphonucleotides, the C-terminal domain of DnaK protein changes in tertiary structure in such a way that fewer hydrophobic segments are exposed on the surface of the protein. After ATP hydrolysis, the number of hydrophobic segments on the surface of the protein is further reduced, and moreover the tertiary structure of the N-terminal domain of the protein changes. These data are discussed in terms of structural and functional relationships of both DnaK and DnaK756 proteins.


Functional delineation of the Ca(2+)-deficient EF-hand in cardiac muscle, with genetically engineered cardiac-skeletal chimeric troponin C.

Cardiac and fast skeletal isoforms of TnC each comprise four putative EF-hand (helix-loop-helix) motifs as potential Ca(2+)-binding sites (sites 1-4), except that site 1 in cardiac TnC is deficient in Ca2+ coordination. In skeletal TnC, the N-terminal sites 1 and 2 are both essential for the trigger mechanism of the contraction switch. However, the mechanism in cardiac muscle is unsettled; it is obscure whether the cardiac site 1 is functionally inert due to calcium deficiency and consequently site 2 is the lone trigger site, or whether sites 1 and 2 perform interactively despite the impairment. These possibilities were addressed by mutagenizing site 1 in skeletal TnC to mimic the cardiac response. In one mutant (STnC-1), two selected Ca(2+)-ligands were abolished. In another (C1/S chimera), 41 N-terminal residues from cardiac TnC were spliced to STnC. The Ca(2+)-binding capacities as well as skinned fiber responses were measured. The STnC-1 derivative failed to switch on contraction. In contrast, the chimeric construct expressed close to full contractile potential in myocardium (74 +/- 3% Po; Po = maximal tension) and also the manifest cardiac phenotype. By devising supplemental chimeric constructs, cardiac-type N-terminal overhang together with cardiac-type EF-hand for site 1 both were found essential for the phenotype. We conclude that cardiac TnC site 1 is actively engaged in the trigger mechanism and in fact dominates the phenotype despite the inability to chelate Ca2+. The N-terminal overhang also participates in this mechanism, which is a novel finding. The conclusion that a non-chelating site functions interactively with a proximal site in cardiac TnC may have wider significance, inasmuch as similar pairings of disparate EF-hands are of common occurrence.


Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis.

Subtilin and nisin are gene-encoded antibiotic peptides that are ribosomally synthesized by Bacillus subtilis and Lactococcus lactis, respectively. Gene-encoded antibiotics are unique in that their structures can be manipulated by mutagenesis of their structural genes. Although subtilin and nisin share considerable structural homology, subtilin has a greater tendency than nisin to undergo spontaneous inactivation. This inactivation is a accompanied by chemical modification of the dehydroalanine at position 5 (DHA5) with a kinetic first-order t1/2 of 0.8 days. It was hypothesized that the R group carboxyl of Glu4 in subtilin participates in the chemical modification of the adjacent DHA5. Noting that nisin has Ile at position 4, site-directed mutagenesis was used to change Glu4 of subtilin to Ile, in order to eliminate this carboxyl-group participation. The DHA5 of this mutant subtilin (E4I-subtilin) underwent modification with a t1/2 of 48 days, which is 57-fold slower than natural subtilin, and the rate of loss of biological activity dropped by a like amount. These results suggest that an intact DHA5 is critical for subtilin activity against bacterial spore outgrowth. A double mutant of subtilin, in which the DHA5 residue of E4I-subtilin was mutated to Ala was devoid of detectable inhibition against spore outgrowth. The specific activity of E4I-subtilin was 3-4-fold higher than natural subtilin, suggesting that an increase in the hydrophobicity of the N-terminal end of the molecule enhances activity. These are the first mutants of subtilin that have been reported, and E4I-subtilin is the first example of any lantibiotic whose properties have been improved by mutagenesis. In order to carry out the mutagenesis, a host-vector pair was constructed that permits a deletion replacement in which the natural subtilin gene is replaced by the mutant gene at the normal location in the chromosome. This maintains normal gene dosage and regulatory responses, as well as eliminates ambiguities caused by expression of the normal and mutant genes in the same cell.


Human lipoprotein lipase: the loop covering the catalytic site is essential for interaction with lipid substrates.

Lipoprotein lipase (LPL), a key enzyme which initiates the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins, consists of multiple functional domains which are necessary for normal activity. The catalytic domain of LPL mediates the esterase function of the enzyme but separate lipid binding sites have been proposed to be involved in the interaction of LPL with emulsified lipid substrates at the water-lipid interface. Like pancreatic lipase (PL), LPL contains a surface loop covering the catalytic pocket that may modulate access of the substrate to the active site of the enzyme. Secondary structural analysis of this loop reveals a helix-turn-helix motif with two short amphipathic helices that have hydrophobic moments of 0.64 and 0.68. In order to investigate the role of the loop in the initial interaction of LPL with its substrate, we utilized site-directed mutagenesis to generate eight constructs in which the amphipathic properties of the loop were altered and expressed them in human embryonal kidney-293 cells. Reducing the amphiphilicity without changing the predicted secondary structure of the loop abolished the ability of the lipase to hydrolyze emulsified, long chain fatty acid triglycerides (triolein) but not the water soluble substrate tributyrin. Replacing the loop of LPL with the loop of hepatic lipase, which differs in 15 of 22 amino acids but is also amphiphilic, led to the expression of an enzyme that retained both triolein and tributyrin hydrolyzing activity. Substitution of the LPL loop by a short four amino acid peptide, which may allow more direct access to the active site than the 22 amino acid loop, enhanced hydrolysis of short chain fatty acid triglycerides by more than 2-fold, while the ability to hydrolyze emulsified substrates was abolished. Thus, disruption of the amphipathic structure of the LPL loop selectively decreases the hydrolysis of emulsified lipid substrate without affecting the esterase or catalytic function of the enzyme. These studies establish that the loop with its two amphipathic helices is essential for hydrolysis of long chain fatty acid substrate by LPL providing new insight into the role of the LPL loop in lipid-substrate interactions. We propose that the interaction between the lipoprotein substrates and the amphipathic helices within this loop may in part determine lipase substrate specificity.


Evidence that 24- and 27-hydroxylation are not involved in the cholesterol-induced down-regulation of hydroxymethylglutaryl-CoA reductase in mouse liver.

Several authors have suggested that 27-hydroxycholesterol may be an important physiological regulator of cholesterol homeostasis. In the present study we investigated the possibility that 24- or 27-hydroxylation of cholesterol is of importance for the down-regulation of hydroxymethylglutaryl (HMG)-CoA reductase in mouse liver induced by dietary cholesterol. Using an accurate method based on isotope dilution-mass spectrometry with deuterated internal standards, we were able to detect significant levels of both 24- and 27-hydroxycholesterol in liver homogenates from normal mice. Feeding cholesterol, 2% for 4 days, increased the levels by 80 and 30%, respectively. No significant hepatic levels of 25-hydroxycholesterol could be demonstrated in untreated mice, and the level of this steroid in cholesterol-treated mice was just above the detection limit. Mouse liver mitochondria were able to catalyze 24- as well as 27-hydroxylation, but not 25-hydroxylation of cholesterol. There was no such conversion in liver microsomes. When using 24-2H2- or 23,23,24,24,25-2H5-labeled cholesterol as substrate a kinetic isotope effect of about 4.5 was observed for the mitochondrial 24-hydroxylation. When using 26,26,26,27,27,27-2H6-labeled cholesterol as substrate a kinetic isotope effect of about 2.5 was observed for the 27-hydroxylation. Use of those deuterium-labeled cholesterol species thus allowed a specific suppression of the rate of 24- and 27-hydroxylation. Feeding mice with 0.05% unlabeled pure cholesterol in the diet for 24 h inhibited the hepatic HMG-CoA reductase activity by about 50%. The same degree of suppression was obtained after feeding with 23,23,24,24,25-2H5- and 26,26,26,27,27,27-2H6-labeled cholesterol. Were mitochondrial 24- and 27-hydroxylation of importance, one would expect reduced suppression of HMG-CoA reductase when feeding deuterated cholesterol, due to the isotope effects. As this was not the case, it is concluded that neither 24-hydroxylation nor 27-hydroxylation are critical for the cholesterol-induced down-regulation of HMG-CoA reductase in mouse liver.


Inhibition of agonist-induced activation of phospholipase C following poxvirus infection.

Recent studies indicate that viruses may influence polyphosphoinositide levels. This study has examined the effects of vaccinia virus infection on phospholipase C activity. Infection of BS-C-1 cells, an African Green Monkey kidney cell line, or A431 cells, a human carcinoma cell line, with vaccinia virus inhibits receptor-mediated phospholipase C activation. As a consequence, agonist-mediated Ca2+ mobilization in BS-C-1 cells also was inhibited by vaccinia virus infection. Alleviation of the inhibition of phospholipase C activation was observed in vaccinia virus-infected cells treated with cycloheximide without influencing uninfected cells. Treatment of infected cells with alpha-amanitin, an inhibitor of host mRNA synthesis but not virus mRNA synthesis, failed to alleviate the inhibition of phospholipase C activation. Together these results suggest that a virus-encoded gene product mediates the inhibition of phospholipase C activation without the need of a virus-induced host factor. Analysis of the processes involved in the formation of inositol (1,4,5)-trisphosphate and mobilization of intracellular Ca2+ indicate that the vaccinia virus gene product exerts its inhibitory effects at the level of phospholipase C activity. This may occur by either directly reducing the amount of phospholipase C, reducing the specific activity of phospholipase C, or by inhibiting the association of phospholipase C with its substrate, phosphatidylinositol 4,5-bisphosphate.


NMR constrained solution structures for laminin peptide 11. Analogs define structural requirements for inhibition of tumor cell invasion of basement membrane matrix.

Peptide 11, CDPGYIGSR-NH2, is a segment of laminin which blocks tumor cell invasion. A high affinity laminin receptor in tumor cells is thought to be blocked by the carboxyl-terminal YIGSR, and conformational energy calculations suggest that the glycine in YIGSR allows an important conformational bend. We replaced the YIGSR glycine residue in peptide 11 with either D-alanine or L-alanine to allow or disfavor the proposed glycine bend. We found the Gly7-->D-Ala7 analog to be equal to peptide 11 in inhibiting tumor cell invasion of basement membrane matrix. The Gly7-->L-Ala7 analog was much less capable of invasion inhibition. Two-dimensional 1H-1H NMR was used to study the solution conformations of the peptide 11 analogs. NOESY experiments revealed close NH-NH contacts in peptide 11 and the D-Ala7 analog, but not in the L-Ala7 analog. Molecular dynamics generated low energy structures with excellent NOE agreement for peptide 11 and its analogs. Both peptide 11 and the D-Ala7 analog, but not the less active L-Ala7 analog, were predicted to have similar bends around Gly7 or D-Ala7. These results suggest that a bend in the YIGSR region of peptide 11 may be important for the binding of laminin to its metastasis-associated receptor.


Aplysia peptide neurotransmitters beta-bag cell peptide, Phe-Met-Arg-Phe-amide, and small cardioexcitatory peptide B are rapidly degraded by a leucine aminopeptidase-like activity in hemolymph.

We have been investigating the role of proteolytic enzymes in the inactivation of peptide neurotransmitters in the marine snail Aplysia. Previous studies (Squire, C. R., Talebian, M., Menon, J. G., Dekruyff, S. D., Lee, T. D., Shively, J. E., and Rothman, B. S. (1991) J. Biol. Chem. 266, 22355-22363) showed that neuroactive fragments of the neurotransmitter alpha-bag cell peptide (alpha-BCP) were rapidly degraded (t1/2 = 0.5-2.7 min) in plasma, hemolymph that had been cleared by centrifugation. Degradation was caused by one or more enzymes resembling mammalian leucine amino-peptidase (LAP, EC 3.4.11.1). In this report we show that three other Aplysia peptide neurotransmitters, beta-BCP(1-5) (Arg-Leu-Arg-Phe-His), FMRFa (Phe-Met-Arg-Phe-amide), and SCPB(1-9) (Met-Asn-Tyr-Leu-Ala-Phe-Pro-Arg-Met-amide) are rapidly degraded (t1/2 = 0.3-2.4 min) in plasma by apparently the same LAP-like enzyme(s). Our findings strongly suggest that the LAP-like enzyme(s), by means of its broad substrate specificity and access to the extracellular spaces of the nervous system in vivo, plays a significant role in the inactivation of many Aplysia peptide neurotransmitters, and they raise the possibility that proteolytic enzymes in the extracellular fluid contribute significantly to the inactivation of peptide neurotransmitters in other animal species.


Ferroxidase kinetics of horse spleen apoferritin.

Protein ferroxidase site(s), which catalyze the reaction between ferrous ion and dioxygen, have long been thought to play a role in core formation in ferritin; however, the mechanism of the reaction has never been studied in detail. In the present work, the enzymatic activity of ferritin was examined using oximetry, the net Fe2+ oxidation reaction being as follows. [formula: see text] The reaction exhibits saturation kinetics with respect to both Fe2+ and O2 (apparent Michaelis constants: Km,Fe = 0.35 +/- 0.01 mM and Km,O2 = 0.14 +/- 0.03 mM). The enzyme has a turnover number kcat = 80 +/- 3 min-1 at 20 degrees C with maximal activity at pH 7. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe(II) oxidation occurs in 1-electron steps. Zinc(II) is a competitive inhibitor of iron(II) oxidation at Zn2+/apoprotein ratios > or = 6 (inhibitor constant KI,Zn = 0.067 +/- 0.011 mM) but appears to be a noncompetitive inhibitor at lower ratios (< or = 2), indicating the presence of more than one type of zinc binding site on the protein. At increments of 50 Fe2+/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. However, when larger increments are employed, some iron oxidation appears to occur on the surface of the mineral core. The results of these studies emphasize the role of the protein shell in all phases of core growth and confirm the presence of a functionally important catalytic site in ferritin in addition to other binding sites on the protein for iron.


Rat hepatonuclear factor PS-1 regulates tissue-specific activity of the S14 promoter in vitro.

We have previously identified a rat hepatonuclear factor, PS-1 that binds to the thyroid hormone responsive gene, S14. To determine whether PS-1 is involved in regulating tissue-specific expression of the S14 gene, we have correlated the DNA binding activity of PS-1 with mRNA-S14 expression in a variety of tissues. Gel retardation analysis revealed a pattern of binding to the recognition site that was characteristic of tissues with high levels of mRNA-S14, a different pattern was found in tissues which do not express the gene. Competition studies using mutant oligonucleotides showed that the first 4 nucleotides and the CAAT motif contained within the PS-1 recognition sequence are essential for protein binding. C/EBP, a CCAAT-transcription factor binds to the PS-1 recognition site thus raising the possibility that both C/EBP and PS-1 may belong to the same family of proteins. Next we used a cell-free transcription assay to measure activity of a template, pS14-GFC(-72), that contained the PS-1 sequence. The pS14-GFC(-72) template was active in hepatonuclear extracts but deletion of or competition with the PS-1 binding sequence rendered the construct inactive. A template containing three PS-1 binding sequences increased S14 promoter activity by 12- to 13-fold. In nuclear extracts from spleen and testis, relative S14 promoter activity was only 2% of that in the liver, this observation mimicked closely in vivo expression of the gene. Mixing together extracts from liver and spleen in varying proportions, prior to incubation with S14 template, yielded a linear increase in S14 promoter activity that correlated with the amount of liver extract in the reaction. This finding is consistent with the absence of an essential factor or factors in spleen that is/are required for S14 promoter activity in vitro. In summary, PS-1 binds to a DNA sequence that contains a CAAT motif and appears to play a critical role in determining tissue-specific activity of the S14 promoter in vitro.


Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase contains two stable phosphorylation sites, Thr-197 and Ser-338 (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). Thr-197 is very resistant to dephosphorylation and thus cannot typically be autophosphorylated in vitro once the stable subunit is formed. Ser-338 is slowly dephosphorylated and can be rephosphorylated autocatalytically. In addition to these two stable phosphorylation sites, a new site of autophosphorylation, Ser-10, was identified. Phosphorylation at Ser-10 does not have a major effect on activity, and phosphates from Ser-10 or Ser-338 are not transferred to physiological substrates such as the type II regulatory subunit. Autophosphorylation at Ser-10 is associated with one of the two major isoelectric variants of the catalytic subunit. The form having the more acidic pI can be autophosphorylated at Ser-10 while the more basic form of the catalytic subunit cannot. Phosphorylation at Ser-10 does not account for the two isoenzyme forms. Since the reason for two isoelectric variants of the catalytic subunit is still unknown, it is not possible to provide a structural basis for the difference in accessibility of Ser-10 to phosphorylation. Either Ser-10 is not accessible in the more basic form of the catalytic subunit or some other type of post- or cotranslational modification causes Ser-10 to be a poor substrate. Whether the myristoyl group at the amino-terminal Gly is important for Ser-10 autophosphorylation remains to be established. The isoenzyme forms of the catalytic subunit do not correspond to the gene products coded for by the C alpha and C beta genes.


Oxidation-reduction properties of Escherichia coli thioredoxin reductase altered at each active site cysteine residue.

Thioredoxin is a small oxidation-reduction (redox) mediator protein. Its reduction by NADPH is catalyzed by the flavoenzyme thioredoxin reductase. Site-directed mutagenesis has provided forms of the reductase in which Cys135 and Cys138 have each been changed to a serine residue (Prongay, A. J., Engelke, D. R., and Williams, C. H., Jr. (1989) J. Biol. Chem. 264, 2656-2664). Cys135 and Cys138 form the redox-active disulfide in the oxidized enzyme. The redox properties of the two altered forms of Escherichia coli thioredoxin reductase have been determined from pH 6.0 to 9.0. Photoreduction of TRR(Ser135,Cys138) produces the blue, neutral semiquinone species, which disproportionates (Kf = 0.73) to an apparent maximum of 29% of the total enzyme as the semiquinone. In contrast, the semiquinone formed on TRR(Cys135,Ser138) during a photoreductive titration does not disproportionate and 70% of the enzyme is stabilized as the semiquinione. Reductive titrations have demonstrated that 1 mol of sodium dithionite (2 electrons)/mol of FAD is required to fully reduce TRR(Ser135,Cys138) whereas 2 mol of dithionite/mol of FAD are required to fully reduce TRR(Cys135,Ser138). The oxidation-reduction midpoint potentials for the 1-electron and 2-electron reductions of TRR(Ser135,Cys138) have been determined by NADH/NAD+ titrations in the presence of a mediator, benzyl viologen. The midpoint potential for the 2-electron reduction of TRR(Ser135,Cys138) is -280 mV, at pH 7.0 and 20 degrees C. Thus, the redox potential is similar to that of the FAD/FADH2 couple in the dithiol form of wild type enzyme, -270 mV (corrected to 20 degrees C) (O'Donnell, M. E., and Williams, C. H., Jr. (1983) J. Biol. Chem. 258, 13795-13805). The delta Em/delta pH is -57.1 mV, which corresponds to a proton stoichiometry of 2 H+/2 e-.A maximum of 19% of the enzyme forms a stable semiquinone species during the titration, and the potentials for the oxidized enzyme/semiquinone couple, E2, and the semiquinone/reduced enzyme couple, E1, are -306 and -256 mV, respectively, at pH 7.0 and 20 degrees C. These studies provide evidence that the residue at position 138 exerts a greater effect on the FAD than does the residue at position 135.


Post-translational processing of an O-glycosylated protein, the human CD8 glycoprotein, during the intracellular transport to the plasma membrane.

The biosynthesis of human CD8 glycoprotein in transfected rat epithelial cells produces an unglycosylated precursor, an intermediate species only initially O-glycosylated, and a doublet mature form carrying neutral and sialylated O-linked oligosaccharides with the core-2 structure (Pascale, M. C., Malagolini, N., Serafini-Cessi, F., Migliaccio, G., Leone, A., and Bonatti, S. (1992) J. Biol. Chem. 267, 9940-9947). In this study the most relevant post-translational events: dimerization, addition of the first O-linked GalNAc, fulfillment of O-linked chains, as well as expression of involved glycosyltransferases, have been examined and correlated with the localization and transit rate of CD8 through the exocytic pathway. The glycosyltransferase activities measured in rat epithelial cells transfected with human CD8 DNA are entirely consistent with the primary structure assigned to CD8 oligosaccharides. The half-time of appearance of the initially O-glycosylated precursor and mature form was estimated to be 4 and 14 min, respectively, and the half-time for delivery of mature CD8 to the cell surface was found to be about 30 min, indicating a very fast routing. Pulse experiments with [35S]cysteine at 37 degrees C followed by chase-periods at low temperatures showed that folding/dimerization occurs before routing to the Golgi apparatus, whereas the addiction of O-linked GalNAc appears to take place later, very likely in cis-Golgi cisternae. Treatment of cells with monensin accumulated the intermediate CD8 form carrying non-elongated O-linked GalNAc, whereas brefeldin A treatment produced a sialylated glycoprotein species with a mobility faster than the mature CD8. These results indicate that the two drugs affect assembly of O-linked chains at different time of their processing.


Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.


Osteogenic protein-2. A new member of the transforming growth factor-beta superfamily expressed early in embryogenesis.

Osteogenic protein-2, OP-2, a new member of the transforming growth factor-beta (TGF-beta) superfamily, closely related to the osteogenic/bone morphogenetic proteins, was discovered in mouse embryo and human hippocampus cDNA libraries. The TGF-beta domain of OP-2 shows 74% identity to OP-1, 75% to Vgr-1, and 76% to BMP-5, hence OP-2 may also have bone inductive activity. The genomic locus of OP-2 has seven exons, like OP-1, and spans more than 27 kilobases (kb). In the C-terminal TGF-beta domain, OP-2 has a unique additional cysteine. Mouse embryos express relatively high levels of OP-2 mRNA at 8 days, two species of 3 and 5 kb. A careful study of mRNA expression of the osteogenic proteins in specific organs revealed discrete mRNA species for BMP-3, BMP-4, BMP-5, and BMP-6/Vgr-1 in lung or liver of young and adult mice. OP-1 is expressed in kidney; however, OP-2 and BMP-2 mRNAs were not detected in any organs studied, suggesting an early developmental role.


Structure and expression of neutrophil gelatinase cDNA. Identity with type IV collagenase from HT1080 cells.

Neutrophils synthesize and store intracellularly a 92-kDa type IV collagenase (gelatinase), the primary structure of which is unknown. We designed a primer based on the highly conserved cysteine-switch region of metalloproteinases and employed the polymerase chain reaction to generate a probe of the human neutrophil gelatinase (HNG) gene. This probe was used to clone the cDNA encoding HNG by screening a chronic granulocytic leukemia cDNA library. In vitro translation of the cDNA-derived HNG mRNA yielded a major product of 78 kDa and smaller autolytically activated or degraded products, all of which were recognized by anti-HNG antibody. The HNG cDNA sequence is nearly identical to that encoding a 92-kDa gelatinase secreted by HT1080 cells. In addition, primer extension and S1 analysis reveal that the above two gelatinase transcripts have similar initiation sites. The HNG cDNA hybridized to a 2.8-kilobase mRNA from chronic granulocytic leukemia cells. HNG mRNA expression was absent from uninduced HL60 cells and from HL60 cells induced to granulocytic maturation with Me2SO. However, unlike other neutrophil secondary granule genes, HNG mRNA was detected in HL60 cells induced to monocytic maturation with 12-O-tetradecanoylphorbol 13-acetate. This suggests that the HNG gene may be subject to differential control pathways in two related but distinct hematopoietic lineages.


Palmitylation of neuromodulin (GAP-43) is not required for phosphorylation by protein kinase C.

Neuromodulin (also designated GAP-43, B-50, and F-1) is a prominent protein kinase C substrate attached to the membranes of neuronal growth cones during development and to presynaptic membranes in discrete subsets of adult synapses. In this study, we have examined the relationship between the attachment of neuromodulin to membranes and its phosphorylation by protein kinase C. To address this issue, we have compared wild-type and mutant neuromodulins expressed in cells that normally lack the protein. Wild-type neuromodulin expressed in Chinese hamster ovary cells was associated with membranes, incorporated [3H]palmitic acid, and was phosphorylated in response to phorbol ester treatment. Substitution of serine 41, the in vitro protein kinase C site, abolished the phorbol ester response, indicating that serine 41 serves as the sole protein kinase C phosphorylation site in vivo. Substitution of the putative fatty acylation sites, cysteines 3 and 4, abolished membrane association as well as [3H]palmitic acid labeling of neuromodulin. Fatty acylation therefore appears to serve as the mechanism for anchoring neuromodulin to membranes. Surprisingly, the soluble cysteine substitution mutant was phosphorylated by protein kinase C at a rate indistinguishable from that of the wild-type protein. Therefore, membrane association may not be required for the phosphorylation of neuromodulin by protein kinase C.


Interconversion of GRP78/BiP. A novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor.

Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.


The role of the mature domain of proOmpA in the translocation ATPase reaction.

The export of proOmpA, the precursor of outer membrane protein A from Escherichia coli, requires preprotein translocase, which is comprised of SecA, SecY/E, and acidic phospholipids. Previous studies of proOmpA translocation intermediates (Schiebel, E., Driessen, A. J. M., Hartl, F.-U., and Wickner, W. (1991) Cell 64, 927-939) suggested that the "slippage" of the translocating polypeptide chain and the high level of ATP hydrolysis, characteristic of the "translocation ATPase," were part of a futile cycle. To examine the role of the mature domain of proOmpA in its translocation-dependent ATP hydrolysis, we used chemical cleavage to generate NH2-terminal fragments of this preprotein. Each fragment contained the 21-residue leader region and either 53 or 228 residues of the mature domain (preproteins P74 and P249, respectively). As observed with full-length proOmpA, the translocation of each fragment requires ATP and both the SecA and SecY/E domains of translocase and is stimulated by the transmembrane proton electrochemical gradient. The apparent maximal velocities of P74 and proOmpA translocation are similar. While the translocation of P74 and of proOmpA show the same apparent Km for ATP, far less ATP is hydrolyzed during the translocation of P74. Thus, the mature carboxyl-terminal domain of proOmpA has a major role in supporting the translocation ATPase.