Genetic and biochemical analysis of in vivo protein folding and subunit assembly.
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Biomedical subjects
Publications and source records attributed to J King.
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The effect of supernatants of concanavalin A-activated spleen cells (CAS) on the expression of various antigens, especially Ia antigens, on BALB/c B lymphoid cells, was examined. This study demonstrates the following: (i) CAS enhanced the expression of Ia antigens on four out of five BALB/c lymphoid cell lines. (ii) CAS selectively modulates the expression of Ia and H-2D, but not sIgM or viral gp70 expression, on X16C 8.5 tumor cells. The enhanced levels of Ia expression on B lymphoid tumor cells were also detected by using anti-Ia monoclonal antibodies. (iii) The molecular weight of soluble factor(s) affecting Ia and H-2 was approximately 40,000 estimated by gel filtration on a Sephadex G-200 column. (iv) Type 1 interferon but not interleukin 1, interleukin 2, or T-cell-replacing factor enhanced the expressions of Ia and H-2D antigens. (v) The activity of CAS-modulating Ia and H-2 antigens was eliminated by acidic treatment. It was concluded from this study that at least one of the factor(s) in CAS, modulating the antigenic expression of B-lymphoid cells, was interferon-like in nature. From our findings, a possible immunoregulatory mechanism by interferon was suggested: T cells, after stimulation of mitogens or antigens, secrete interferons which modulate the expression of Ia and H-2 on B cells. Then B cells, whose Ia and H-2 were modulated selectively by T-soluble factors(s), might interact with T cells much more efficiently.
Eighteen months' use of DSM-III by a consultation-liaison service in a university hospital is reported. DSM-III's multiaxial system seems to be feasible and revealing, but presents some problems. Adjustment Disorder and Organic Mental Disorders were the most frequent Axis I diagnoses. Approximately one-fourth of the patients received an Axis II diagnosis and 85% received an Axis III diagnosis. In 70% of the patients there was both a psychiatric diagnosis (on Axis I or II) and a physical disorder (on Axis III). On Axis IV and V, this population appeared to be under moderate to severe psychosocial stress and had, as a group, a fair or better level of previous adaptive functioning. The possible significance of these findings and potential areas for further refinements and investigations are discussed.
Temperature-sensitive mutations in the gene encoding the trimeric tail spike protein of phage P22 interfere with protein maturation at 39 degrees C. We show here that temperature-sensitive mutations at many sites block the folding pathway prior to accumulation of the partially folded protrimer intermediate. Temperature-shift experiments indicate that at least some of the mutants accumulate an earlier intermediate in the folding pathway. Immunoprecipitation experiments suggest that the conformation of the isolated temperature-sensitive polypeptide chains is closer to that of the unfolded chain than to that of the mature spike formed at permissive temperature. The sites of these mutations probably represent amino acid sequences that play key roles during the folding of the tail spike polypeptide chain but are not important in the mature protein.
The caries-preventive effects of two toothpastes were tested in a 3-year clinical trial involving 1319 children aged 11-13 years. The test toothpastes were: 1) a low abrasion paste containing 0.8% sodium monofluorophosphate (MFP), and 2) a paste containing 3% sodium trimetaphosphate (TMP). The control toothpaste was a conventional, moderately abrasive paste containing 0.8% sodium monofluorophosphate. The children were examined clinically and radiologically each year. Toothpaste was supplied to the children's homes, and its use was unsupervised. Caries increments after 3 years, in terms of DMF teeth and DMF surfaces, showed no significant differences between the MFP test group (444 children) and the MFP control group (450 children). However, the TMP test group (425 children) had significantly higher caries increments than the MFP control group. All three groups of children showed some improvement in oral hygiene and in gingival health, but there were no significant differences between the groups.
A comparison has been made of intestinal fluid absorption between male Okamoto spontaneously hypertensive rats (s.h.r.) and normotensive male Wistar controls. S.h.r. show enhanced fluid absorption both in hypertensive adults and in young s.h.r. before hypertension has developed. Several potential causes for increased fluid transport in s.h.r. were tested using pharmacological antagonists. It is unlikely that enhanced fluid absorption is due to high sympathetic nervous activity, the renin-angiotensin system or is secondary to hypertension. Intestine from s.h.r. have a high short-circuit current indicating a change in ion pump activity. These results are discussed in relation to the possible causes of increased fluid (ion) transport by the intestine of s.h.r.
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One of the most difficult problems confronting occupational therapists, teachers, and parents in dealing with latency-age learning-disabled children is handling oppositional behavior. Pre-adolescent boys, in particular, frequently have difficulty working cooperatively in a group, relating to peers, staying with a task, and seeing the consequences of their actions. This paper provides a rationale for treatment of children in groups and describes the development and early results of an activity group approach to counseling learning-disabled children with behavior problems using Gazda's developmental model. Group members showed improved ability to relate with siblings and peers; more mature and cooperative behavior; greater independence and impulse control; and increased verbal expression of anger. Parent and teacher feedback on behavior changes was positive.
For the study of the protein--protein and protein--nucleic acid interactions in the assembly of virus particles, laser Raman spectra have been obtained in H2O and D2O solutions and as a function of temperature for the following Salmonella phage P22 components: mature phage particles, isolated mature phage DNA, mature protein shells empty of DNA, precursor protein shells (procapsids), and purified coat, scaffolding and tail-spike proteins. The spectra confirm that the condensed DNA within the phage capsid assumes the B-form secondary structure similar to aqueous DNA and reveal no evidence of specific molecular interactions between subgroups of DNA and protein subunits of the phage capsid. No differences were detected in the highly irregular secondary structure of the major capsid protein in mature capsids, empty capsids (lacking DNA), procapsids, and empty procapsids (lacking scaffolding protein). Features of both primary and secondary structures of the viral scaffolding and tail-spike proteins are also revealed by the spectra. Differences in thermal stability of tyrosyl side-chain interactions were observed between scaffolding protein extracted from the procapsid and within the procapsid. These differences correspond to different hydrogen bonding configurations of p-hydroxyphenyl groups and provide indirect evidence for the participation of the scaffolding proteins in specific macromolecular interactions within the procapsid.
As part of a genetic analysis of the in vivo folding and subunit assembly of the P22 tail spike endorhamnosidase, we have studied the maturation of the newly synthesized 76,000-dalton polypeptide chains into thermostable tail spike oligomers. Four of 15 temperature-sensitive mutations in the structural gene for this protein result in electrophoretically distinct tail spikes. Cells mixedly infected with wild type and an electrophoretic variant produce two hybrid species, with mobilities intermediate between the parental species, indicating that the native tail spike is a trimer. Mature trimers are resistant to denaturation by sodium dodecyl sulfate (SDS): at room temperature the trimer migrates in an SDS gel as if it were not binding significant amounts of SDS, whereas the heat-denatured chain migrates as expected of an SDS-polypeptide complex. The mature trimer is also resistant to trypsin digestion. Lysates of infected cells contain SDS and trypsin-sensitive forms of the newly synthesized tail spike polypeptide chains. These are probably incompletely or incorrectly folded chains. SDS and trypsin resistance were used to measure the efficiency of in vivo folding and subunit assembly of the mature trimer from its polypeptide chains. This decreased from 90% at 27 degrees C to only 15% at 42 degrees C. These results are consistent with the existence or a labile intermediate or step in the folding or subunit assembly of the thermostable tail spike protein. We discuss the possibility that the achievement of certain structural features of mature proteins may entail difficulties in their folding pathways.
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The intestinal parasite Giardia lamblia was found in the stools of three patients with retinal arteritis. Iridocyclitis was also present in two of these patients, who had not responded to treatment with systemic corticosteroids. Antiparasitic drug treatment was followed by improvement of ocular and systemic findings.
Newly synthesized tail spike polypeptide chains mature from trypsin- and NaDodSO4-sensitive unfolded chains to trypsin- and NaDodSO4-resistant native trimers with a t1/2 of 5 min at 30 degrees C. A metastable intermediate in subunit folding and assembly was trapped by chilling and isolated by electrophoresis through nondenaturing gels in the cold. A fraction of the intermediate could be matured into native trimers in vitro by incubating at physiological temperature. Mixing experiments with electrophoretically distinct mutant proteins showed that the precursor that matured in vitro represented three tail spike polypeptide chains already associated with each other but not fully folded. Identification of this intermediate reveals that the processes of polypeptide chain folding and subunit assembly are coupled in this large structural protein.
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We describe a 14-month-old female who presented with hepatomegaly 1 month after abdominal trauma. Eight years later, radiographic and histologic studies confirmed the diagnosis of Budd--Chiari syndrome with evolution to cirrhosis.