[SMARCA4-deficient undifferentiated carcinoma of the gastrointestinal tract: a clinicopathological and immunohistochemical study of nine cases].

Objective: To investigate the clinicopathological features, immunophenotype and differential diagnoses of SMARCA4-deificient undifferentiated carcinoma (SMARCA4-DUC) of the gastrointestinal tract. Methods: The clinicopathological data and immunohistochemical profiles of nine cases of SMARCA4-DUC of the gastrointestinal tract diagnosed in Fudan University Shanghai Cancer Center, from 2018 to 2021, were analyzed retrospectively. The relevant literature was reviewed. Results: There were seven males and two females with age at presentation ranging from 39 to 74 years (mean 58 years, median 64 years). The tumor occurred in the stomach (6 cases), right hemicolon (2 cases) and duodenum (1 case). The main symptoms included dysphagia, abdominal pain, diarrhea and melena. Five cases were resected, and the tumor sizes ranged from 5.0 to 8.7 cm (mean 6.7 cm). Microscopically, the tumor was composed of sheets of undifferentiated round to epithelioid cells with large vesicular nuclei harboring prominent nucleoli and displaying brisk mitotic activity. Foci of dyscohesive rhabdoid cells were also noted. The tumor cells were generally uniform; however, prominent pleomorphism and spindle cell component was present in one case each. Five cases contained areas of coagulative necrosis, and one case showed myxoid change of the stroma. By immunohistochemistry, eight cases showed complete loss of BRG1 (SMARCA4) and BRM (SMARCA2) expression. Whereas the expression of these two markers was lost in the epithelioid component of one case, it remained in the spindle cell component (mosaic pattern). Apart from one case with partial expression of pan-cytokeratin, all other eight cases showed either limited (<5%, n=5) or totally negative (n=3) staining of pan-cytokeratin. In addition, four cases also expressed CD34, SOX2 and SALL4. Six patients had follow-up data: four died of disease within 1 year. Conclusions: SMARCA4-DUC of the gastrointestinal tract represents a highly aggressive malignancy with poor outcome. Due to lack of cell-specific differentiation, it is not uncommonly misdiagnosed as a wide variety of poorly-differentiated or undifferentiated tumors. Increased recognition of this rare but distinctive entity not only facilitates the diagnosis and differential diagnosis, but also provides important therapeutic and prognostic information for the clinicians.

[The impact of donor-to-recipient gender compatibility on outcomes of haploid hematopoietic stem cell transplantation in patients with hematological malignancies].

Objective: To investigate how gender differences between the donor and the recipient affect the effectiveness of antithymocyte globulin (ATG) and pure peripheral blood stem cell (PBSC) hematopoietic stem cell transplantation (haplo-HSCT) in the treatment of malignant hematological diseases. Methods: From February 2015 to September 2020, 648 hematological malignancies patients underwent myeloablative condition regimen haplo-HSCT treatment at the Bone Marrow Transplant Center of the First Affiliated Hospital of Zhejiang University. The median age was 32 (14-62) years, with 363 males (56.0% ) and 285 females (44.0% ) present. 242 cases of acute lymphoblastic leukemia (ALL) (37.3% ) , 293 cases of acute myeloid leukemia (AML) (45.2% ) , 56 cases of myelodysplastic syndrome (MDS) (8.7% ) , 27 cases of non-Hodgkin's lymphoma (NHL) (4.2% ) , and 30 cases of other hematological malignancies (4.6% ) . Results: ① The 3-year overall survival (OS) , DFS, the incidence of Ⅱ-Ⅳ grade acute graft-versus-host disease (aGVHD) , the incidence of Ⅲ-Ⅳ grade aGVHD, the 3-year incidence of moderate & severe chronic GVHD (cGVHD) , severe cGVHD, the 3-year incidence of relapse, and NRM of the whole group were (73.10±1.90) % , (70.80±1.90) % , (33.96±1.87) % , (13.08±1.33) % , (35.10±2.14) % , (10.66±1.38) % , (19.43±1.67) % , and (9.80±1.24) % , respectively. ②There was no statistically significant difference between the donor-recipient gender match and donor-recipient gender mismatch groups in the 28-day cumulative neutrophil engraftment rate, 28-day cumulative platelet engraftment rate, the incidence of Ⅱ-Ⅳ grade aGVHD, the incidence of Ⅲ-Ⅳ grade aGVHD, 3-year OS, 3-year DFS, the cumulative incidence of relapse, NRM, and incidence of moderate & severe cGVHD, severe cGVHD. ③The 28-day cumulative neutrophil engraftment rate did not differ statistically between the male-female, female-female, male-male, and female-male groups (P=0.148) . The incidence of Ⅱ-Ⅳ grade aGVHD, the incidence of Ⅲ-Ⅳ grade aGVHD, 3-year OS, 3-year DFS, cumulative relapse rate, and NRM, and the incidence of cGVHD were not statistically different among the four groups (P>0.05) . The 28-day cumulative platelet engraftment rate of the female-male group was significantly lower than male-female group, and the female-female group [ (91.45±2.63) % vs. (94.77±1.75) % , P=0.004; (91.45±2.63) % vs. (95.54±2.05) % , P=0.005]. No significant difference existed in the 28-day cumulative platelet engraftment rate between the female-male group and the male-male group [ (91.45±2.63) % vs. (95.08±1.41) % , P=0.284]. ④Among patients ≤35 years old, the 3-year incidence of severe cGVHD patients receiving sister donors and sibling donors were (26.71±5.90) % and (10.33±4.43) % , respectively (P=0.054) . Patients accepting daughter donors and son donors had a 3-year incidence of moderate and severe cGVHD that was 40.07% vs. 27.41% , respectively, among those over 35 (40.07±6.65) % vs. (27.41±4.54) % (P=0.084) . ⑤Female donors to male recipients had a significantly lower 28-day cumulative platelet engraftment rate compared to the other groups [ (91.45±2.63) % vs. (95.08±0.95) % , P=0.037]. ⑥ Female donors to male recipients had a significantly lower 28-day cumulative platelet engraftment rate than the other groups in the ATG-Fresenius (ATG-F) 10 mg/kg group [ (89.29±4.29) % vs. (94.49±1.45) % , P=0.037]. But when compared to the other groups in the Rabbit Antihuman Thymocyte Immunoglobulin (rATG-T) 6 mg/kg group, the 28-day cumulative platelet implantation rate between female donors and male recipients was not significantly different [ (93.44±3.38) % vs. (95.62±1.26) % , P=0.404]. Conclusion: The main clinical outcomes of patients with malignant blood diseases following transplantation are unaffected by the gender combination of the donor and patient in the haplo-HSCT mode based on ATG and PBSC sources. Female donors to male recipients have a lower 28-day cumulative platelet engraftment rate and longer platelet engraftment times.

[Application of pulse contour cardiac output monitoring technology in fluid resuscitation of severe burn patients in shock period].

Objective: To investigate the application of pulse contour cardiac output (PiCCO) monitoring technology in fluid resuscitation of severe burn patients in shock period. Methods: From January 2015 to December 2019, 33 patients with severe burns who were hospitalized in Guangzhou Red Cross Hospital, meeting the inclusion criteria, were recruited into a retrospective cohort study with their clinical information collected. The patients were divided into PiCCO monitoring group with 15 cases (13 males and 2 females, aged (43±13) years) and routine monitoring group with 18 cases (14 males and 4 females, aged (39±9) years) according to the monitoring method used. After admission, all the patients were rehydrated following the rehydration formula of the Third Military Medical University for shock period. In routine monitoring group, the fluid resuscitation of patients was performed by monitoring indicators such as urine volume and blood pressure, while PiCCO monitoring was performed among patients in PiCCO monitoring group, and their fluid resuscitation was guided by the patient's condition and the hemodynamic parameters (without pursuing normal levels of the parameters) of PiCCO monitoring on the basis of normal monitoring indicators in routine monitoring group. The colloids coefficients, the electrolyte coefficients (compared with the corresponding rehydration formula value of 0.75 mL·kg(-1)·% total body surface area (TBSA)(-1) of the Third Military Medical University for shock period during the first 24 h post injury), the total rehydration coefficients, and the urine volumes during the first and second 24 h post injury, the lactic acid level, the base excess level, and the oxygenation index at admission and 24, 48 h after admission, and the mechanical ventilation time, the wound healing time, and the death ratio of patients in the two groups were recorded. The cardiac index, the global end-diastolic volume index (GEDVI), the intrathoracic blood volume index (ITBVI), the extravascular lung water index (EVLWI), and the systemic vascular resistance index (SVRI) of patients in PiCCO monitoring group at post injury hour 24, 48, and 72 and the abnormal cases were recorded. Data were statistically analyzed with Fisher's exact probability test, independent-sample or one-sample t test, analysis of variance for repeated measurement, and Bonferroni correction. Results: During the first 24 h post injury, the colloids coefficients of patients in PiCCO monitoring group was (0.69±0.15) mL·kg(-1)·%TBSA(-1), which was significantly less than (0.85±0.16) mL·kg(-1)·%TBSA(-1) in routine monitoring group (t=-2.612, P<0.05). Compared with the rehydration formula value of the Third Military Medical University for shock period, only the colloids coefficient of patients in routine monitoring group during the first 24 h post injury was significantly increased (t=2.847, P<0.05). There were no statistically significant differences between the two groups in the colloids coefficients of patients during the second 24 h post injury, or the electrolyte coefficients, the total rehydration coefficients, the urine volumes of patients during the first and the second 24 h post injury (t=0.579, -0.011, 0.417, -1.321, -0.137, 0.031, 1.348, P>0.05). The lactic acid level, the base excess level, the oxygenation index of patients at admission and 48 h after admission, and the oxygenation index of patients at 24 h after admission between the two groups were similar (t=-1.837, 0.620, 0.292, -1.792, 1.912, -0.167, 1.695, P>0.05). The levels of lactic acid and base excess of patients in PiCCO monitoring group were (4.8±1.4) and (1.2±5.5)mmol/L, respectively, which were significantly better than (7.0±1.5) and (-2.8±3.0) mmol/L in routine monitoring group at 24 h after admission (t=-3.904, 2.562, P<0.05 or P<0.01). There were no statistically significant differences between the two groups in the mechanical ventilation time or the wound healing time of patients (t=-0.699, -0.697, P>0.05), or the death ratio of patients (P>0.05). In PiCCO monitoring group, the GEDVI, and the ITBVI of patients were lower than the normal low values at post injury hour 24 and 48, which were in the normal range at post injury hour 72; the cardiac index of patients increased gradually and recovered to normal at post injury hour 48; the SVRI of patients increased significantly at post injury hour 24 and then gradually decreased to normal; the EVLWI average of patients at all time points post injury were less than 10 mL/kg. At post injury hour 24, most of the hemodynamic parameters of more than or equal to 8/15 patients in PiCCO monitoring group were abnormal, and the abnormal proportion decreased later. Conclusions: On the basis of traditional monitoring indicators, the use of PiCCO monitoring technology combined with the patient's condition (without pursuing normal levels of the parameters) in guiding the fluid resuscitation in severe burn patients can reduce the usage of colloid and better improve tissue perfusion, with the resuscitation effect being better than conventional monitoring.

Common characteristics shared by different differential phase contrast imaging methods.

There are many variations of differential phase contrast imaging methods. Although these imaging methods are different in configuration, they are alike in imaging by extracting differential phase information through the evaluation of the refraction angles. In this paper, we investigate common characteristics shared by various different differential phase contrast imaging methods.

A new iterative algorithm to reconstruct the refractive index.

The latest developments in x-ray imaging are associated with techniques based on the phase contrast. However, the image reconstruction procedures demand significant improvements of the traditional methods, and/or new algorithms have to be introduced to take advantage of the high contrast and sensitivity of the new experimental techniques. In this letter, an improved iterative reconstruction algorithm based on the maximum likelihood expectation maximization technique is presented and discussed in order to reconstruct the distribution of the refractive index from data collected by an analyzer-based imaging setup. The technique considered probes the partial derivative of the refractive index with respect to an axis lying in the meridional plane and perpendicular to the propagation direction. Computer simulations confirm the reliability of the proposed algorithm. In addition, the comparison between an analytical reconstruction algorithm and the iterative method has been also discussed together with the convergent characteristic of this latter algorithm. Finally, we will show how the proposed algorithm may be applied to reconstruct the distribution of the refractive index of an epoxy cylinder containing small air bubbles of about 300 micro of diameter.

Dopamine and glutamate induce distinct striatal splice forms of Ania-6, an RNA polymerase II-associated cyclin.

Control of neuronal gene expression by drugs or neurotransmitters is a critical step in long-term neural plasticity. Here, we show that a gene induced in the striatum by cocaine or direct dopamine stimulation, ania-6, is a member of a novel family of cyclins with homology to cyclins K/T/H/C. Further, different types of neurotransmitter stimulation cause selective induction of distinct ania-6 isoforms, through alternative splicing. The longer Ania-6 protein colocalizes with nuclear speckles and is associated with key elements of the RNA elongation/processing complex, including the hyperphosphorylated form of RNA polymerase II, the splicing factor SC-35, and the p110 PITSLRE cyclin-dependent kinase. Distinct types of neuronal stimulation may therefore differentially modulate nuclear RNA processing, through altered transcription and splicing of ania-6.

Reconstitution studies using the helical and carboxy-terminal domains of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

Enzyme I of the bacterial phosphoenolpyruvate:sugar phosphotransferase system can be phosphorylated by PEP on an active-site histidine residue, localized to a cleft between an alpha-helical domain and an alpha/beta domain on the amino terminal half of the protein. The phosphoryl group on the active-site histidine can be passed to an active-site histidine residue of HPr. It has been proposed that the major interaction between enzyme I and HPr occurs via the alpha-helical domain of enzyme I. The isolated recombinant alpha-helical domain (residues 25-145) with approximately 80% alpha-helices as well as enzyme I deficient in that domain [EI(DeltaHD)] with approximately 50% alpha-helix content from M. capricolum were used to further elucidate the nature of the enzyme I-HPr complex. Isothermal titration calorimetry demonstrated that HPr binds to the alpha-helical domain and intact enzyme I with = 5 x 10(4) and 1.4 x 10(5) M(-)(1) at pH 7.5 and 25 degrees C, respectively, but not to EI(DeltaHD), which contains the active-site histidine of enzyme I and can be autophosphorylated by PEP. In vitro reconstitution experiments with proteins from both M. capricolum and E. coli showed that EI(DeltaHD) can donate its bound phosphoryl group to HPr in the presence of the isolated alpha-helical domain. Furthermore, M. capricolum recombinant C-terminal domain of enzyme I (EIC) was shown to reconstitute phosphotransfer activity with recombinant N-terminal domain (EIN) approximately 5% as efficiently as the HD-EI(DeltaHD) pair. Recombinant EIC strongly self-associates ( approximately 10(10) M(-)(1)) in comparison to dimerization constants of 10(5)-10(7) M(-)(1) measured for EI and EI(DeltaHD).

Autophosphorylation of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system requires dimerization.

Enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system undergoes a slow monomer-dimer transition. In vitro autophosphorylation of Enzyme I by PEP was studied at limiting concentrations of the protein. Addition to incubation mixtures containing wild-type Enzyme I of inactive or low-activity mutant forms of Enzyme I resulted in stimulation of autophosphorylation activity. The kinetics of the activation fit well to a model in which the active form of Enzyme I is the dimer. These experiments provide support for the argument that only the dimeric form of Enzyme I can be autophosphorylated.

Topography of the interaction of HPr(Ser) kinase with HPr.

The phosphocarrier protein, HPr, from Gram-positive organisms and mycoplasmas is a substrate for an ATP-dependent kinase that phosphorylates serine 46. In Gram-negative organisms, the corresponding HPr is not phosphorylated on serine 46 and the ATP-dependent kinase is absent. To determine the specificity requirements for phosphorylation of Mycoplasma capricolum HPr, a chimera in which residues 43-57 were replaced by the Escherichia coli sequence was constructed. The chimeric protein folded properly, but was not phosphorylated on either serine 46 or histidine 15. A dissection of the region required for phosphorylation specificity was carried out by further mutagenesis. The deficiency in phosphorylation at histidine 15 was localized primarily to the region including residues 51-57. Activity studies revealed that residues 48, 49, and 51-53 are important for recognition of M. capricolum HPr by its cognate HPr(Ser) kinase. The characteristics of this region suggest that the kinase-HPr interaction occurs mainly through a hydrophobic region. Molecular modeling comparisons of M. capricolum HPr and the chimeric construct provided a basis for interpreting the results of the activity assays.

A promiscuous binding surface: crystal structure of the IIA domain of the glucose-specific permease from Mycoplasma capricolum.

BACKGROUND: The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a bacterial and mycoplasma system responsible for the uptake of some sugars, concomitant with their phosphorylation. The sugar-specific component of the system, enzyme II (EII),consists of three domains, EIIA, EIIB and EIIC. EIIA and ELLB are cytoplasmic and EIIC is an integral membrane protein that contains the sugar-binding site. Phosphoenolpyruvate (PEP) provides the source of the phosphoryl group, which is transferred via several phosphoprotein intermediates, eventually being transferred to the internalized sugar. Along the pathway, EIIA accepts a phosphoryl group from the phosphocarrier protein HPr and transfers it to EIIB. The structure of the glucose-specific EIIA (EIIAglc) from Mycoplasma capricolum reported here facilitates understanding of the nature of the interactions between this protein and its partners. RESULTS: The crystal structure of EIIAglc from M. capricolum has been determined at 2.5 A resolution. two neighboring EIIAglc molecules associate with one another in a front-to-back fashion, such that Glu149 of one molecule forms electrostatic interactions with the active-site histidine residues, His90 and His75, of the other. Glu149 is therefore considered to mimic the interaction that a phosphorylated histidine of a partner protein makes with EIIA. Another interaction, an ion pair between the active-site Asp94 and Lys168 of a neighboring molecule, may be analogous to the interaction between Asp94 of EIIAglc and Arg17 of HPr. Analysis of molecular packing in this crystal, and in the crystals of two other homologous proteins from Escherichia coli and Bacillus subtilis, reveals that in all cases active-site hydrophobic residues are involved in crystal contacts, but in each case a different region of the neighboring molecule is involved. The transition-state complexes of M. capricolum EIIAglc with HPr and EIIBglc have been modeled; in each case, different structural units are shown to interact with EIIAglc. Many of the interactions are hydrophobic with no sequence specificity. The only specific interaction, other than that formed by the phosphoryl group, involves ion pairs between two invariant aspartate residues of EIIAglc and arginine/lysine residues of HPr or EIIBglc. CONCLUSIONS: The non-discriminating nature of the hydrophobic interactions that EIIAglc forms with a variety of partners may be a consequence of the requirement for interaction with a variety of proteins that show no sequence or structural similarity. Nevertheless, specificity is provided by an ion-pair interaction that is enhanced by the apolar nature of the interface.

Expression, purification, and characterization of enzyme IIA(glc) of the phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum.

The gene encoding enzyme IIA(glc) (EIIA) of the phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum was cloned into a regulated expression vector. The purified protein product of the overexpressed gene was characterized as an active phosphoacceptor from HPr with a higher pI than previously described EIIAs. M. capricolum EIIA was unreactive with antibodies directed against the corresponding proteins from either Gram-positive or Gram-negative bacteria. Enzyme IIA(glc) behaved as a homogeneous, monomeric species of 16,700 Mr in analytical ultracentrifugation. The circular dichroism far-UV spectrum of EIIA reflects a low alpha-helical content and predominantly beta-sheet structural content: temperature-induced changes in ellipticity at 205 nm showed that the protein undergoes reversible, two-state thermal unfolding with Tm = 70.0 +/- 0.3 degrees C and a van't Hoff deltaH of 90 kcal/mol. Enzyme I (64,600 Mr) from M. capricolum exhibited a monomer-dimer-tetramer association at 4 and 20 degrees C with dimerization constants of log K(A) = 5.6 and 5.1 [M(-1)], respectively, in sedimentation equilibrium experiments. A new vector, capable of introducing an N-terminal His tag on a protein, was developed in order to generate highly purified heat-stable protein (HPr). No significant interaction of EIIA with HPr was detected by gel-filtration chromatography, intrinsic tryptophanyl residue fluorescence changes, titration calorimetry, biomolecular interaction, or sedimentation equilibrium studies. While Escherichia coli EIIA inhibits Gram-negative glycerol kinase activity, the M. capricolum EIIA has no effect on the homologous glycerol kinase. The probable regulator of sugar transport systems, HPr(Ser) kinase, was demonstrated in extracts of M. capricolum and Mycoplasma genitalium. Gene mapping studies demonstrated that, in contrast to the clustered arrangement of genes encoding HPr and enzyme I in E. coli, these genes are located diametrically opposite in the M. capricolum chromosome.

Sequence and organization of genes encoding enzymes involved in pyruvate metabolism in Mycoplasma capricolum.

The region of the genome of Mycoplasma capricolum upstream of the portion encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed open reading frames corresponding to numerous genes involved with the oxidation of pyruvate. The deduced gene organization is naox (encoding NADH oxidase)-lplA (encoding lipoate-protein ligase)-odpA (encoding pyruvate dehydrogenase EI alpha)-odpB (encoding pyruvate dehydrogenase EI beta)-odp2(encoding pyruvate dehydrogenase EII)-dldH (encoding dihydrolipoamide dehydrogenase)-pta (encoding phosphotransacetylase)-ack (encoding acetate kinase)-orfA (an unknown open reading frame)-kdtB-ptsI-crr. Analysis of the DNA sequence suggests that the naox and lplA genes are part of a single operon, odpA and odpB constitute an additional operon, odp2 and dldH a third operon, and pta and ack an additional transcription unit. Phylogenetic analyses of the protein products of the odpA and odpB genes indicate that they are most similar to the corresponding proteins from Mycoplasma genitalium, Acholeplasma laidlawii, and Gram-positive organisms. The product of the odp2 gene contains a single lipoyl domain, as is the case with the corresponding proteins from M. genitalium and numerous other organisms. An evolutionary tree places the M. capricolum odp2 gene product in close relationship to the corresponding proteins from A. laidlawii and M.genitalium. The dldH gene encodes an unusual form of dihydrolipoamide dehydrogenase that contains an aminoterminal extension corresponding to a lipoyl domain, a property shared by the corresponding proteins from Alcaligenes eutrophus and Clostridium magnum. Aside from that feature, the protein is related phylogenetically to the corresponding proteins from A. laidlawii and M. genitalium. The phosphotransacetylase from M. capricolum is related most closely to the corresponding protein from M. genitalium and is distinguished easily from the enzymes from Escherichia coli and Haemophilus influenzae by the absence of the characteristic amino-terminal extension. The acetate kinase from M. capricolum is related evolutionarily to the homologous enzyme from M. genitalium. Map position comparisons of genes encoding proteins involved with pyruvate metabolism show that, whereas all the genes are clustered in M. capricolum, they are scattered in M. genitalium.

Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity.

The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr). The isolated amino-terminal domain (EIN) of E. coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr. An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein. A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield. Intact E. coli EI is effective in phosphotransfer from PEP to HPr from E. coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum. Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E. coli or M. capricolum requires the intermediacy of HPr. The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E. coli, B. subtilis, and M. capricolum as well as to EIAglc from E. coli. These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr.

Structural evidence for the evolutionary divergence of mycoplasma from gram-positive bacteria: the histidine-containing phosphocarrier protein.

BACKGROUND: The three-dimensional structures of histidine-containing phosphocarrier protein (HPr), a member of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), have been determined from Gram-negative and Gram-positive bacteria. The structure of HPr reported here for Mycoplasma capricolum is the first protein structure to be determined for this class of organism. Comparative structural studies with the bacterial proteins highlight sequence-structure correlations relevant to proposals about the evolutionary origin of mycoplasmas. RESULTS: The crystal structure of HPr from M. capricolum has been determined and refined at 1.8 A resolution, revealing the same overall fold as that of other HPrs of known structure. However, mycoplasma HPr resembles HPrs from Gram-positive bacteria more closely than those from Gram-negative bacteria. As in HPrs from Bacillus subtilis and Escherichia coli, the phosphoryl group carrier (His15) forms the N-terminal cap of a helix, but in contrast to the other crystal structures, the side chain of the adjacent Arg17 is conformationally disordered. A sulfate ion interacts with Ser46, a residue known to be phosphorylated in a regulatory manner. CONCLUSIONS: The greater degree of structural similarity of the M. capricolum HPr to HPrs from Gram-positive rather than Gram-negative bacteria is consistent with the proposal that mycoplasma evolved from Gram-positive bacteria. The proposal that no major conformational transition is required for phosphorylation of the active-site histidine is reinforced by comparing the crystal structures with and without an anion in the active site. The conformational disorder of the Arg17 side chain suggests that its guanidinium group does not have to form specific interactions with other protein groups before phosphorylation at His15. The association of a sulfate ion with Ser46 serves as a model for HPr(Ser46-P). As there is no evidence of a conformational change accompanying Ser46 phosphorylation, the inhibitory effect of this event may be attributable to altered surface electrostatics.

Overproduction and purification of the Mycoplasma capricolum phosphocarrier protein, HPr, of the phosphoenolpyruvate: sugar phosphotransferase system.

The gene (ptsH) for the phosphocarrier protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system from Mycoplasma capricolum was previously cloned and sequenced. We present here the results of experiments in which the ptsH gene was cloned into a vector for high level expression in Escherichia coli of the phosphocarrier protein. Conditions were developed for overproduction and purification of HPr by a two-column procedure. The purified protein, analyzed by Edman degradation and mass spectrometry, was found to have been processed by removal of the N-terminal methionine residue. Examination of the purified protein by gel electrophoresis under isoelectric focusing conditions revealed that it has an unusually high isoelectric point.

Unique dicistronic operon (ptsI-crr) in Mycoplasma capricolum encoding enzyme I and the glucose-specific enzyme IIA of the phosphoenolpyruvate:sugar phosphotransferase system: cloning, sequencing, promoter analysis, and protein characterization.

The region of the genome of Mycoplasma capricolum encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed a unique arrangement of the pts operon. In all other bacterial species characterized thus far, the gene encoding Enzyme I (ptsI) in the pts operon is located immediately downstream of the gene (ptsH) encoding HPr, a general energy coupling protein of the PTS. In M. capricolum, ptsH and ptsI reside on 2 distinct operons at separate loci on the chromosome (Zhu PP, Reizer J, Reizer A, Peterkofsky A, 1993, J Biol Chem 268:26531-26540). In the present work, it is shown that the Mycoplasma Enzyme I gene is preceded by an open reading frame homologous to the product of the Escherichia coli kdtB gene and is followed by the gene (crr) encoding Enzyme IIAglc. Northern blot analysis indicated that ptsI and crr constitute a dicistronic operon that includes an independent promoter for the crr gene. Primer extension studies established the transcription start sites for the ptsI and crr genes. The products of the ptsI and crr genes are homologous to previously sequenced Enzymes I and IIAglc proteins but are more similar to the counterpart proteins from gram-positive than to those from gram-negative organisms. The deduced amino acid sequence of the Mycoplasma Enzyme I shows that it differs from other Enzymes I by having fewer acidic amino acids and more basic, amidated, and aromatic amino acids. The deduced amino acid sequence of the Mycoplasma Enzyme IIAglc indicates that it is the shortest (154 residues) of the proteins in this class and it is the only Enzyme IIAglc with a tryptophan and a cysteine residue. In vitro sugar phosphorylation studies with extracts from E. coli and Bacillus subtilis and purified proteins indicated that the Mycoplasma HPr is not a phosphoacceptor from the E. coli Enzyme I, whereas the Mycoplasma Enzyme IIAglc accepts and transfers phosphate from both E. coli and B. subtilis PTS components.

Unique monocistronic operon (ptsH) in Mycoplasma capricolum encoding the phosphocarrier protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system. Cloning, sequencing, and characterization of ptsH.

The region of the genome of Mycoplasma capricolum encompassing the gene (ptsH) encoding HPr, a general energy-coupling protein of the phosphoenolpyruvate:sugar phosphotransferase system, was cloned and sequenced. Examination of the sequence revealed a unique arrangement of the ptsH gene. In all other bacterial species characterized thus far, the ptsH gene is part of a polycistronic operon that includes the gene (ptsI) encoding Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system; the M. capricolum ptsH gene is part of a monocistronic operon that is situated between two open reading frames unrelated to phosphoenolpyruvate:sugar phosphotransferase system function. The gene immediately upstream of ptsH codes for a helicase, and the open reading frame immediately downstream of ptsH, although not homologous to any previously identified protein, contains a signature sequence characteristic of [C-5] cytosine-specific DNA methylases. The product of the ptsH gene has characteristics similar to the HPr protein produced by Gram-positive organisms: it has a greater sequence similarity to HPrs of Gram-positive bacteria than to those of Gram-negative organisms, it is phosphorylated by a protein kinase derived from Gram-positive organisms, and it complements sugar phosphorylation activity in Gram-positive extracts. The high calculated isoelectric point (pI = 9.18) and the absence of glutamate residues in the C-terminal region distinguish the M. capricolum HPr from all previously described HPrs.

Effect of L-4-oxalysine on ultrastructures of liver cells in mice.

Mice were given ig L-4-oxalysine (I-677) 10, 50, and 100 mg.kg-1.d-1 for 7 d. On d 8 the hepatocytes showed accumulation of lipid droplets followed by loss of matrices in cytoplasm. The total area of lipid droplets was far less than 25% of mean section of hepatocytes. The injury of mitochondria and RER was only found in the groups of medium and high dose. The lipidoses and regional topolysis of cytoplasm graduated away at same pace. After 4 wk the hepatocytes were restored to normal. Such finding suggests that the site of action of I-677 be at the cytoplasmic ground substance. The inhibition of protein synthesis causes a decrease in albumin carrier, that may be the main mechanism of steatosis of liver cells induced by I-677.