Non-histone chromosomal proteins easily extractible from chick erythrocytes.

Those non-histone chromosomal proteins which are easily extractible from chick erythrocytes differ substantially from proteins similarly extracted from other tissues of various species. Although a protein P1 was isolated along with histone H1 by extraction of calf thymus chromatin with HC1O4, the same procedure did not extract this protein from chick erythrocyte chromatin of either normal or regenerating blood. Likewise , non-histone proteins extracted with 0.35 M NaCl from calf thymus differed from those of normal chick erythrocytes, which were qualitatively identical but quantitatively inferior to those of regenerating blood. The major protein of about 25 000 molecular weight, totally extracted with 0.35 M NaCl from calf thymus, was not found in chick erythrocyte chromatin, but rather another major protein of about 35 000 molecular weight was partially extracted from erythrocytes.

Location of the globular region in chicken erythrocyte histone H5.

Chicken erythrocyte histone H5 has been cleaved by acetic acid hydrolysis at the two aspartic acid residues 65 and 99 and the 4 peptides (1-65), (66-185) (1-99) (100-185) recovered in a pure form. 270 MHz magnetic resonance and circular dichroic studies show that the two C-terminal peptides are unable to form secondary or tertiary structure. The N-terminal peptides however, form both secondary and tertiary structure. In particular, the peptide (1-99) at high ionic strength possesses a similar number of helical residues to intact histone H5 and also had a closely related nuclear magnetic resonance spectrum. It is concluded that the peptide (1-99) contains most, but not quite all of the residues that are included in the globular segment of histone H5.

Heat stress preconditioning does not protect renal epithelial Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters from their modulation by severe heat stress.

This study compares the effects of heat and osmotic stress on heat stress protein (HSP) production while examining the putative protective action of HSPs on modulation of Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters in Madin-Darby canine kidney (MDCK) epithelial cells by severe heat stress (46 degrees C, 15 min). Preconditioning heat stress (43 degrees C, 20 min) followed by 4 h recovery at 37 degrees C led to a 35-fold increase of HSP70 mRNA expression measured by Northern blot analysis. The protein content of HSP70 and HSP27, assessed by Western blots, was augmented by 5- and 2-fold, respectively, after 6 h of recovery. In contrast to preconditioning heat stress, hyperosmotic stress (520 vs. 320 mosm) elevated HSP70 mRNA content only by 7-fold and did not significantly affect the protein content of HSP70 or HSP27. Neither cell survival, assessed as lactate dehydrogenase (LDH) release, nor the basal activities of the ion transporters and their modulation by protein kinase C, P(2)-purinoceptor and cell volume were altered by preconditioning heat stress. Severe heat stress increased extracellular LDH content from 3+/-2 to 23+/-5% and enhanced Na(+),K(+),Cl(-) and Na(+),P(i) cotransport activity by 2-3-fold. The volume- and protein kinase C-dependent regulation of these carriers was abolished by severe heat stress while regulation by P(2)-purinoceptors was preserved. Preconditioning heat stress diminished severe heat stress-induced LDH release to 11+/-4% but did not protect Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters from activation by severe heat stress and did not prevent severe heat stress-induced inactivation of protein kinase C- and volume-dependent signaling pathways. These results show that in MDCK cells, preconditioning heat stress-induced HSPs are not involved in the regulation of Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters and do not protect them from modulation by severe heat stress.

Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.

Comparison between histones FV and F2a2 of chicken erythrocyte. I. Structure, stability and conformation of the free proteins.

Purified chicken erythrocyte histones FV and F2a2 were studied by means of circular dichroism as a function of ionic strength and temperature. The percentage of alpha-helical regions was calculated by comparison with reference spectra obtained with four standard proteins of known tertiary structure. Maximal alpha-helical organization, reached in high ionic strength, was estimated to 14% and 23% for FV and F2a2 respectively. We have compared our experimental determinations of the secondary structure of F2a2 with predictions made from amino-acid sequence according to Fasman's rules. When instability induced by the presence of charged residues close together is taken into account, a good agreement is found between predicted and observed values. The thermal denaturation of FV is cooperative and, unlike F2a2, seems to obey a two-state transition. The classical Arrhenius plot is linear, which indicates that the heat capacity is the same in both the native and the denatured state. Such a behaviour is typical of an expanded configuration of FV even in the "native" state.

Drosophila stretchin-MLCK is a novel member of the Titin/Myosin light chain kinase family.

Members of the titin/myosin light chain kinase family play an essential role in the organization of the actin/myosin cytoskeleton, especially in sarcomere assembly and function. In Drosophila melanogaster, projectin is so far the only member of this family for which a transcription unit has been characterized. The locus of another member of this family, a protein related to Myosin light chain kinase, was also identified. The cDNA and genomic sequences published explain only the shorter transcripts expressed by this locus. Here, we report the complete molecular characterization of this transcription unit, which spans 38 kb, includes 33 exons and accounts for transcripts up to 25 kb in length. This transcription unit contains both the largest exon (12,005 nt) and the largest coding region (25,213 nt) reported so far for Drosophila. This transcription unit features both internal promoters and internal polyadenylation signals, which enable it to express seven different transcripts, ranging from 3.3 to 25 kb in size. The latter encodes a huge, titin-like, 926 kDa kinase that features two large PEVK-rich repeats, 32 immunoglobulin and two fibronectin type-III domains, which we designate stretchin-MLCK. In addition, the 3' end of the stretchin-MLCK transcription unit expresses shorter transcripts that encode 86 to 165 kDa isoforms of stretchin-MLCK that are analogous to vertebrate Myosin light chain kinases. Similarly, the 5' end of the Stretchin-Mlck transcription unit can also express transcripts encoding kettin and Unc-89-like isoforms, which share no sequences with the MLCK-like transcripts. Thus, this locus can be viewed as a single transcription unit, Stretchin-Mlck (genetic abbreviation Strn-Mlck), that expresses large, composite transcripts and protein isoforms (sequences available at http://www.academicpress.com/jmb), as well as a complex of two independent transcription units, the Stretchin and Mlck transcription units (Strn and Mlck, respectively) the result of a "gene fission" event, that encode independent transcripts and proteins with distinct structural and enzymatic functions.

Protection against necrosis but not apoptosis by heat-stress proteins in vascular smooth muscle cells: evidence for distinct modes of cell death.

We have reported previously that cultured vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR) show higher proliferation and cell death than normotensive controls. In addition to protecting cells against death, heat stress proteins (HSPs) appear to play a role in cell proliferation. This investigation examines the involvement of HSP72 and HSP27 in altered SHR VSMC proliferation and death. We have performed detailed discriminatory analysis to characterize which type of VSMC death is induced by heat stress (HS) and serum deprivation. Serum deprivation induced apoptosis (caspase-3 cleavage and DNA laddering) and secondary necrosis, the 2 processes being a continuum of each other. In contrast, acute HS (46 degrees C, 30 minutes), which inhibited BN. lx and SHR VSMC proliferation by 2-fold, increased necrosis (by 5-fold and 2-fold, respectively) but not apoptosis. HSP72 and HSP27 expression evoked in VSMC by mild HS (44 degrees C, 15 minutes) 6 hours before acute HS prevented the inhibition of proliferation and induction of necrosis with no effect on serum deprivation-induced or staurosporine-induced apoptosis. This induced expression of HSP72 and HSP27 did not eliminate the higher basal proliferation, apoptosis, and necrosis of SHR VSMC compared with BN.lx VSMC, suggesting that these HSPs are not involved in altered SHR VSMC proliferation and death. Also, although apoptosis and necrosis may be a continuum, in VSMC the 2 processes may be distinguished by HS, in which only necrosis is prevented by prior HSP accumulation. This observation may be of use in designing strategies for cellular protection.

Binding of GM1-ganglioside to a synthetic peptide derived from the lysosomal sphingolipid-activator-protein saposin B.

Saposin B is a lysosomal sphingolipid-activator-protein which activates GM1-ganglioside hydrolysis by lysosomal beta-galactosidase. To identify the structural elements of saposin B implicated in sphingolipid binding, we studied a synthetic peptide corresponding to a predicted alpha-helix, sapB-18, spanning residues 52 to 69 of saposin B. The circular dichroism spectrum of sapB-18 at pH 4.4 was consistent with a 44% alpha-helix content. As shown by intrinsic Tyr fluorescence studies of sapB-18, this peptide binds the GM1-ganglioside with a Kd of about 7 microM. Thus, we suggest that a putative amphipathic alpha-helix between residues 52 and 69 of saposin B plays a major role in the recognition and binding of GM1-ganglioside by saposin B.

Binding of GM1 ganglioside to a synthetic peptide derived from the lysosomal sphingolipid activator protein saposin B.

Saposin B is a lysosomal sphingolipid activator protein which activates GM1 ganglioside hydrolysis by lysosomal beta-galactosidase. To identify the structural elements of saposin B implicated in sphingolipid binding, we studied a synthetic peptide corresponding to a predicted alpha-helix, sapB-18, spanning residues 52-69 of saposin B. The circular dichroism spectrum of sapB-18 at pH 4.4 was consistent with a 44% alpha-helix content. As shown by intrinsic Tyr fluorescence studies of sapB-18, this peptide binds the GM1 ganglioside with a Kd of about 7 microM. Thus, we suggest that a putative amphipathic alpha-helix between residues 52 and 69 of saposin B plays a major role in the recognition and binding of GM1 ganglioside by saposin B.

Complete cDNA sequences of mouse rod photoreceptor cGMP phosphodiesterase alpha- and beta-subunits, and identification of beta'-, a putative beta-subunit isozyme produced by alternative splicing of the beta-subunit gene.

We have characterized overlapping cDNA clones encoding cGMP phosphodiesterase (PDE) alpha- and beta-subunits of mouse retinal rod photoreceptors. The open reading frames predict an alpha-subunit of 100 kDa (856 residues), and a beta-subunit of 99 kDa (853 residues). Sequence analysis of two of twelve beta-subunit clones predicts the presence in the retina of an additional PDE, termed beta', which is generated by alternative splicing of the beta-subunit gene. beta' differs from beta only at the C-terminus being 55 residues shorter and lacking the Caax motif found at the C-termini of both the alpha- and beta-subunits. A 300 residue segment thought to contain the active site is present in the C-terminal half of alpha, beta and beta'.

Fluorescence anisotropy decay of ethidium bromide bound to nucleosomal core particles.

We have measured the fluorescence anisotropy decay of ethidium bromide bound to nucleosomal core particles (145 DNA base pairs) for very small values of the binding ratio (0.0005 less than or equal to r less than or equal to 0.01). For r = 0.0005 the anisotropy decay could be described by a sum of two exponential functions. The two correlation times theta 1 and theta 2 increase with r until r congruent to 0.0025 and then decrease while the apparent fundamental anisotropy A'0 decreases until r congruent to 0.0025 and then remains constant. The anisotropy decay parameters of the first ethidium molecule bound to a core particle have been obtained by extrapolating theta 1, theta 2 and A'0 to r = 0. We propose the following interpretation of these results. The first bound ethidium molecule is located on a DNA segment linked by its two ends to the histone core. This ethidium molecule follows the torsional motion of the DNA segment. The length of this segment (15 base pairs) was determined by fitting a mathematical expression, derived from the torsional dynamics of DNA, to the extrapolated anisotropy decay. The second ethidium molecule binds to the same DNA segment which explains the decrease of A'0 by fast excitation energy transfer. At the same time theta 1 and theta 2 increase. On binding, a third ethidium molecule breaks the links between the DNA segment and the histone core. This entails the decrease of theta 1 and theta 2.

Predictors of physical outcomes in pediatric bone marrow transplantation.

The purpose of the present study was to investigate the hypothesis that family factors, in conjunction with clinical factors, are associated with physical outcomes in pediatric BMT. A prospective study of 68 pediatric patients (mean age = 7.5 years; ranging from 4 months to 18 years) undergoing BMT was carried out over a 6.5 year period. Physicians rated initial prognosis on a (0-5) scale which incorporated the child's diagnosis, known risk factors, and type of donor. Both parents individually completed two psychometrically sound questionnaires assessing family well-being and marital satisfaction. Cox proportional hazards survival analyses were performed to determine predictors of death (44% of the patients died). Potential predictor variables included were: initial prognosis, type of transplant, patient's age, socioeconomic status, marital satisfaction and family status, and family stress. Initial prognosis, as estimated by the physician, (RR = 0.62, 95% CI = 0.40, 0.97) was the best predictor of survival. Initial clinical factors are clearly critical in outcomes for pediatric BMT patients.

Unrelated bone marrow transplantation for leukocyte adhesion deficiency.

The severe form of leukocyte adhesion deficiency type I (LAD-I) usually leads to death early in life. Allogeneic haematopoietic transplantation is the only cure. Unrelated transplantation has been reported only once. We describe three children with LAD-I transplanted with T cell non-depleted bone marrow from unrelated HLA-matched donors. All patients engrafted, one of them at second transplant. One patient developed grade I and one grade II acute GVHD. Two patients are alive, one of them with a decrease in CD11/CD18 expression. Early referral for HLA-matched unrelated BMT is a reasonable option for patients with LAD-I lacking an HLA-matched related donor.

Intravenous busulfan for allogeneic hematopoietic stem cell transplantation in infants: clinical and pharmacokinetic results.

SUMMARY: High-dose busulfan is an important component of myeloablative regimens. Variable drug exposure may occur following oral administration. Therefore, the use of intravenous busulfan has been advocated. Previous work has suggested a cumulative dosage of 16 mg/kg for haematopoietic transplantation in children less than 3 years of age, but only limited data are available in infants. Pharmacokinetics of intravenous busulfan administered at the suggested dosage were studied in 14 infants (median age 4.7 months). Busulfan plasma concentrations were measured by either GC-MS or HPLC-UV. In seven patients, the dose was decreased to target an area- under- the- curve of 600-1300 micromol min. The median total dose given was 13.8 mg/kg. All patients engrafted. Severe veno-occlusive disease occurred in one patient. Our study demonstrates that a cumulative dosage of 16 mg/kg is associated with higher exposure than expected in infants. We suggest an initial dose of 0.8 mg/kg followed by pharmacokinetically guided dose adjustment.

Results of an unrelated transplant search strategy using partially HLA-mismatched cord blood as an immediate alternative to HLA-matched bone marrow.

Cord blood (CB) is an alternative to other sources of stem cells for transplantation. However, the impact of including CB in the initial strategy of unrelated graft search in a cohort of patients has been the object of limited analysis. Here, we report the results of such a strategy in 91 consecutive children. Absence of mismatch was required for adult donors, and up to two mismatches were allowed for CB grafts, with a nucleated cell dose over 2.5 x 10(7) cells/kg. A graft was found for 84 of the 85 children who remained available for a 3-month search. In all, 64 patients were transplanted, 36 with CB and 28 with bone marrow (BM). Primary graft failure, acute grade II-IV and extensive chronic graft-versus-host disease occurred in five, five and zero CB, and in three, one and two BM patients, respectively. The 3-year survival was 59% in CB and 57% in BM patients. Accepting CB as a source of stem cells offers a graft to almost every child in need of an unrelated transplantation, with a probability of survival similar to that of unrelated BM transplantation.

Multi-site study of incidence of pressure ulcers and the relationship between risk level, demographic characteristics, diagnoses, and prescription of preventive interventions.

OBJECTIVE: To determine the incidence of pressure ulcers in varied populations, and whether demographic characteristics (age, gender, race) and primary diagnosis are factors in pressure ulcer development when the level of risk for developing ulcers is considered. To determine if there is a difference in the type of preventive services prescribed for persons who do or do not develop pressure ulcers when risk is controlled and whether differences can be related to demographic characteristics. DESIGN: Cohort study. SETTING: Two skilled nursing homes, two university operated tertiary care hospitals, and two Veteran's Administration Medical Centers (VAMCs) in Omaha, NE, Durham, NC, and Chicago, IL. PATIENTS: A total of 843 randomly selected patients more than 19 years of age who did not have pressure ulcers on admission to their place of care. Subjects were 63% male, 79% white, and had a mean age of 63 (+/- 16) years. MEASURES: A head-to-toe skin assessment for pressure ulcers recording site and stage of ulcers, scores for the Braden Scale for Predicting Pressure Sore Risk, demographic characteristics (age, sex, race), and primary diagnosis and preventive interventions (turning or repositioning orders and pressure reduction surface) were documented on the patient record. Observations were made every 48 to 72 hours for a minimum of 1 to a maximum of 4 weeks. MAIN OUTCOME MEASURES: Presence/absence and stage of pressure ulcers. MAIN RESULTS: One hundred eight of 843 (12.8%) subjects developed pressure ulcers. The incidence was 8.5%, 7.4%, and 23.9% in tertiary care, VAMCs, and nursing homes, respectively. Logistic regression demonstrated that lower Braden Scale scores, older age and white race predicted pressure ulcers; gender was not predictive. Primary diagnoses were not significant predictors of pressure ulcer risk when the Braden Scale score was entered into the regression. Prescription of turning was predicted by Braden Scale scores and by white race, whereas prescription of pressure reduction was predicted by Braden Scale scores, white race, and female sex. CONCLUSIONS: Risk assessment, rather than diagnoses or demographic characteristics, is recommended as the basis for prescriptive decisions. Risk assessment should cue health care providers to make more judicious use of turning and support surfaces to prevent pressure ulcers. Persons who are at risk for pressure ulcers should have turning and pressure reduction surfaces consistently prescribed and implemented. The costs and goals of preventive prescription for those not at risk for pressure ulcers should be considered.

Cytogenetic analysis of a parachordoma.

We report the cytogenetic and histopathological findings in a 7-year-old female child with an intranasal tumor that is most consistent with a parachordoma. Karyotypic analysis of the tumor revealed clonal numerical and structural chromosome abnormalities. Seven cells displayed recurrent changes: der(2)t(2;4), del(3q), and the loss of chromosomes 9, 10, 20, and 22. Four cells showed a loss of chromosome 17. To the best of our knowledge, these are the first clonal chromosome abnormalities described in parachordoma.

Core particle stability critically depends upon a small number of terminal nucleotides.

An apparently homogeneous population of core particles is in fact composed of three subpopulations which behave differently when exposed to a high concentration of ethidium bromide or to 0.6 M NaCl. These subspecies have been identified by the use of several techniques, viz., electron microscopy, sedimentation velocity and circular dichroism. The electrophoretic analysis of their DNA leads to the conclusion that core particle stability critically depends upon a small number of terminal nucleotides.

Phase II study of carboplatin (CBDCA) in progressive low-grade gliomas.

In this study, the authors sought to investigate the response rate and toxicity of carboplatin in patients with progressive low-grade glioma (LGG). Thirty-two patients with progressive LGG were treated with carboplatin at a dosage of 560 mg/m(2). Treatment was given at 4-week intervals and continued until the disease progressed, unacceptable toxicity supervened, or for 12 additional courses after achieving maximal response. Patients with stable disease were treated with a total of 12 cycles. All patients were treated as outpatients. Patients were evaluated for response to treatment and toxicity. All patients received a minimum of two cycles of carboplatin, and were examined for response. A partial response was achieved in nine patients (28%) and a minimal response in two (6%), for an overall response rate of 34% (11 of 32 patients). Eighteen patients (56%) had stable disease. A partial response was achieved in the nine patients after a median of six cycles (range 4-11 cycles), a minimal response was achieved in the two patients after five cycles. Glioma progression was noted in three patients after three, five, and five cycles, respectively. The 11 patients in whom some response was achieved had either an optic pathway tumor or a juvenile pilocytic astrocytoma. Twenty-six of the 32 patients had those characteristics, making the response rate in that group 42% (11 of 26 patients). Thirty-two patients received a total of 387 cycles of chemotherapy. Hematological toxicity was moderate. Twenty-one patients developed thrombocytopenia (platelet count < 50,000/microl); three patients required one platelet transfusion each. Nine patients developed neutropenia (absolute neutrophil count < 500/microl); one developed fever and required administration of antibiotic agents. One dose adjustment in each of the patients prevented further thrombocytopenia and neutropenia. Two patients with stable disease died of respiratory complications. One patient developed Grade III ototoxicity after receiving five cycles, one patient developed hypersensitivity to carboplatin, and none developed nephrotoxicity. Carboplatin given at a dosage of 560 mg/m(2) every 4 weeks has activity in patients with progressive LGG. This drug regimen is relatively simple and well tolerated. Further investigation and longer follow-up study are warranted.