Functional determinants in transit sequences: import and partial maturation by vascular plant chloroplasts of the ribulose-1,5-bisphosphate carboxylase small subunit of Chlamydomonas.

The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved.

Biogenesis of photosystem II complexes: transcriptional, translational, and posttranslational regulation.

The integral membrane proteins of photosystem II (PS II) reaction center complexes are encoded by chloroplast genomes. These proteins are absent from thylakoids of PS II mutants of algae and vascular plants as a result of either chloroplast or nuclear gene mutations. To resolve the molecular basis for the concurrent absence of the PS II polypeptides, protein synthesis rates and mRNA levels were measured in mutants of Chlamydomonas reinhardtii that lack PS II. The analyses show that one nuclear gene product regulates the levels of transcripts from the chloroplast gene encoding the 51-kD chlorophyll a-binding polypeptide (polypeptide 5) but is not involved in the synthesis of other chloroplast mRNAs. Another nuclear product is specifically required for translation of mRNA encoding the 32-34-kD polypeptide, D1. The absence of either D1 or polypeptide 5 does not eliminate the synthesis and thylakoid insertion of two other integral membrane proteins of PS II, the chlorophyll a-binding polypeptide of 46 kD (polypeptide 6) and the 30-kD "D1-like" protein, D2. However, these two unassembled subunits cannot be properly processed and/or are degraded in the mutants even though they reside in the membrane. In addition, pulse labeling of the nuclear mutants and a chloroplast mutant that does not synthesize D1 mRNA indicates that synthesis of polypeptide 5 and D1 is coordinated at the translational level. A model is presented to explain how absence of one of the two proteins could lead to translational arrest of the other.

NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii.

A precursor (pS) to the small subunit (S) of ribulose1-,5-bisphosphate carboxylase is the major product of cell-free protein synthesis directed by poly(A) containing RNA from Chlamydomonas reinhardtii. We present sequence data for in vitro-synthesized pS, for in vitro-synthesized S that in generated from pS by posttranslational incubation with a Chlamydomonas cell extract, and for in vitro-synthesized, mature S. We show that pS contains an NH2-terminal extension of 44 amino acid residues that is removed by cleavage at the correct site when pS is converted to S by an endoprotease present in the Chlamydomonas cell extract.

Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex.

We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post-translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids. The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls. Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex.

Vision survival after open globe injury predicted by classification and regression tree analysis.

OBJECTIVE: To assist ophthalmologists in treating ocular trauma patients, this study developed and validated a prognostic model to predict vision survival after open globe injury. DESIGN: Retrospective cohort review. PARTICIPANTS: Two hundred fourteen patients who sought treatment at the Wilmer Ophthalmological Institute with open globe injuries from January 1, 2001, through December 31, 2004, were part of the data set used to build the classification tree model. Then, to validate the classification tree, 51 patients were followed up with the goal to compare their actual visual outcome with the outcome predicted by the tree grown from the classification and regression tree analysis. METHODS: Binary recursive partitioning was used to construct a classification tree to predict visual outcome after open globe injury. The retrospective cohort treated for open globe injury from January 1, 2001, through December 31, 2004, was used to develop the prognostic tree and constitutes the training sample. A second independent sample of patient eyes seen from January 1, 2005, through October 15, 2005, was used to validate the prognostic tree. MAIN OUTCOME MEASURES: Two main visual outcomes were assessed: vision survival (range, 20/20-light perception) and no vision (included no light perception, enucleation, and evisceration outcomes). RESULTS: A prognostic model for open globe injury outcome was constructed using 214 open globe injuries. Of 14 predictors determined to be associated with a no vision outcome in univariate analysis, presence of a relative afferent pupillary defect and poor initial visual acuity were the most predictive of complete loss of vision; presence of lid laceration and posterior wound location also predicted poor visual outcomes. In an independent cohort of 51 eyes, the prognostic model had 85.7% sensitivity to predict no vision correctly and 91.9% specificity to predict vision survival correctly. CONCLUSIONS: The open globe injury prognostic model constructed in this study demonstrated excellent predictive accuracy and should be useful in counseling patients and making clinical decisions regarding open globe injury management.

Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly.

The biogenetic interdependence of light-harvesting chlorophyll (Chl) a/b proteins (LHCPs) and antenna pigments has been analyzed for two nuclear mutants of Chlamydomonas that have low levels of Chl b, neoxanthin, and loroxanthin. In mutant PA2.1, the apoprotein precursors (pLHCP II) of the major light-harvesting complex LHC II were synthesized at approximately wild-type rates, processed to their mature size, and rapidly degraded. Because the bulk of labile LHCP II in PA2.1 was soluble, a thylakoid integration factor apparently is defective in this strain. Chl a, Chl b, neoxanthin, and loroxanthin synthesis and accumulation were coordinately reduced in PA2.1, indicating that LHCP II play important regulatory or substrate roles in de novo synthesis of these pigments. Mutant GE2.27 is impaired principally in Chl b synthesis but nonetheless accumulated wild-type levels of all LHCPs. Topology studies of the GE2.27 LHCP II demonstrated that their insertion into thylakoids was incomplete even though they were not structurally altered. Thus, Chl b formation mediates conformational changes of LHCP II after thylakoid integration is initiated. GE2.27 also exhibited very low rates of neoxanthin synthesis and was unable to accumulate loroxanthin. Revertant GE2.27 strains with varying capacities for Chl b formation provided additional evidence that neoxanthin synthesis and accumulation are coupled with the final steps of LHCP II integration into thylakoids. We propose that biogenesis of LHC includes interdependent pigment synthesis/assembly events that occur during LHCP integration into the thylakoid membrane and that defects in these events account for the pleiotropic characteristics of many Chl b-deficient mutants.

Chloroplast RNA Stability in Chlamydomonas: Rapid Degradation of psbB and psbC Transcripts in Two Nuclear Mutants.

Toward understanding regulation of chloroplast transcript abundance, we have isolated and analyzed nuclear mutant strains of Chlamydomonas reinhardtii that lack chloroplast-encoded mRNAs for photosystem II proteins. Mutant 6.2z5 accumulates no transcripts of the psbC locus for the 43-kilodalton chlorophyll-binding protein. In mutant GE2.10, transcripts of psbB, encoding the 47-kilodalton chlorophyll-binding protein, cannot be detected [Jensen, K.H., Herrin, D.L., Plumley, F.G., and Schmidt, G.W. (1986). J. Cell Biol. 103, 1315-1325]. Also, GE2.10 does not accumulate several low molecular weight transcripts from a region of the chloroplast genome proximal to psbB. The levels of mRNAs from other chloroplast genes are not affected in either mutant. Chloroplast transcription was analyzed in permeabilized cells and by in vivo pulse labeling. Although 5[prime] ribonuclease was found as an artifactual activity of permeabilized cells, the results from both assays demonstrated that wild-type levels of psbC transcription occur in mutant 6.2z5 and that chloroplasts of GE2.10 transcribe psbB and adjacent genes. Thus, it appears that the nuclear genes that are mutated in 6.2z5 and GE2.10 encode products that, respectively, confer stability to transcripts from the psbC and the psbB regions of the chloroplast genome.

Isolated progressive visual loss after coiling of paraclinoid aneurysms.

BACKGROUND AND PURPOSE: The proximity of the paraclinoid segment of the internal carotid artery to the visual pathways may result in visual deficits when patients present with aneurysms in this segment. Although surgical clip ligation of these aneurysms has been the standard of care for decades, the advent of coil embolization has permitted endovascular therapy in those aneurysms with favorable dome-to-neck ratios. Although immediate nonprogressive visual loss after coil embolization of paraclinoid aneurysms has been well described, isolated progressive visual loss immediately or shortly following coil embolization, to our knowledge, has not. We have identified 8 patients who experienced progressive loss of vision, unassociated with any other neurologic deficits, developing immediately or shortly after apparently uncomplicated coil embolization of a paraclinoid aneurysm. MATERIALS AND METHODS: This study is a retrospective case series of 8 patients seen at 4 separate academic institutions. Inpatient and outpatient records were examined to determine patient demographics, previous ocular and medical history, and ophthalmic status before endovascular embolization. In addition, details of the primary endovascular therapy and subsequent surgical and nonsurgical interventions were recorded. Follow-up data, including most recent best-corrected visual acuity, postoperative course, and duration of follow-up were documented. RESULTS: Eight patients developed progressive visual loss in 1 or both eyes immediately or shortly after apparently uncomplicated coiling of a paraclinoid aneurysm. MR imaging findings suggested that the visual loss was most likely caused by perianeurysmal inflammation related to the coils used to embolize the aneurysm, enlargement or persistence of the aneurysm despite coiling, or a combination of these mechanisms. Most patients experienced improvement in vision, 2 apparently related to treatment with systemic corticosteroids. CONCLUSION: Patients in whom endovascular treatment of a paraclinoid aneurysm is contemplated should be warned about the potential for both isolated nonprogressive and progressive visual loss in 1 or both eyes. Patients in whom progressive visual loss occurs may benefit from treatment with systemic corticosteroids.

Ultrafast excitation dynamics of low energy pigments in reconstituted peripheral light-harvesting complexes of photosystem I.

Ultrafast dynamics of a reconstituted Lhca4 subunit from the peripheral LHCI-730 antenna of photosystem I of higher plants were probed by femtosecond absorption spectroscopy at 77 K. Intramonomeric energy transfer from chlorophyll (Chl) b to Chl a and energy equilibration between Chl a molecules observed on the subpicosecond time scale are largely similar to subpicosecond energy equilibration processes within LHCII monomers. However, a 5 ps equilibration process in Lhca4 involves unique low energy Chls in LHCI absorbing at 705 nm. These pigments localize the excitation both in the Lhca4 subunit and in LHCI-730 heterodimers. An additional 30-50 ps equilibration process involving red pigments of Lhca4 in the heterodimer, observed by transient absorption and picosecond fluorescence spectroscopy, was ascribed to intersubunit energy transfer.

Immunochemical analysis of baboon (Papio cynocephalus) IgG subclasses.

Few reports are presently available on the existence of IgG subclasses in nonhuman primates. Papain and trypsin digestion of baboon (Papio cynocephalus cynocephalus and P. cynocephalus hamadryas) IgG proteins, in the absence of cysteine, revealed the occurrence of two different protein populations, one being protease-resistant, the other being protease-sensitive. The papain-resistant population is rendered sensitive to digestion upon addition of cysteine. Ion exchange chromatography of the papain-resistant IgG population subsequently demonstrated that it is composed of two different subpopulations varying in their ionic binding affinities. Peptide maps of the Fc fragments of the papain sensitive population and of the Fc fragments of the two papain-resistant subpopulations differing in their binding affinities for ion exchange resins, were different from each other. The biochemical identification of the baboon IgG proteins presented here strongly suggests that they are composed of at least three different subclasses.

Effects of group contingent events upon classroom noise.

The first study investigated a group control procedure for suppression of excessive sound-intensity levels in a regular public school classroom. Reinforcement consisted of a 2-min addition to the class gym period and a 2-min break after maintenance of an unbroken 10-min quiet period as monitored on a decibel meter. Transgressions of the sound limit (42 decibels) resulted in a delay of reinforcement by the resetting of the timer to the full 10-min interval. The results indicated that these procedures were highly effective in suppression and control of sound intensities. The second experiment utilized a similar procedure coupled with a procedure of eliminating out-of-seat behavior. Experiment III studied the effects of Exp. II procedures on a single student's out-of-seat behavior rate. All procedures were found effective.

[The optimal protein quantity in infant nutrition]. / Uber das Optimum an Eiweiss in der Ernährung des jungen Säuglings

Nowadays, the trend of recommendations to prepare infant formulas with only 2 per cent or less of protein does not correspond with the optimal protein demand and protein utilization in infants. Such formulas often prompt mothers to add carbohydrates to the protein half-strength milks in order to satisfy the infant's hunger. This often leads to the unwanted effect to obesity. There is no evidence to prove that infants above the age of 1 month should not receive 2.2 or 2.3% of protein in their formulas. This quantity is fully utilized by the infants; it has no disadvantages and is safe. Hence, the recommendations should not be based on the minimum daily requirements but on the optimal utilization, whereby the tendency to obesity can also be reduced.

[Tuberous sclerosis as cause of neuropathological symptoms allegedly due to smallpox vaccination (author's transl)]. / Begehrte Vakzinations-Schadensanerkenntnis bei einer seltenen (angeborenen) Krankheit

Following smallpox vaccination a patient presented with neuropathological symptoms, the real cause of which was tuberous sclerosis, a congenital disease.

[Detection of a hitherto unknown ninhydrin positive substance in the haemolysate of patients with metachromatic leucodystrophy (author's transl)]. / Nachweis einer bisher unbekannten Ninhydrin-positiven Substanz im Hämolysat von Patienten mit metachromatischer Leukodystrophie

A hitherto unknown substance, which appears between glycine and alanine in ion exchange chromatograms, was found in the erythrocyte cold haemolysate of two children with metachromatic leucodystophy; this substance could not be detected in the haemolysates from patients with other (brain) diseases. It would be worthwhile to test for this symptom in confirmed, still active (florid) cases of metachromatic leucodystrophy.

Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase.

A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylyase (dimerizing), EC 4.1.1.39] has been identified among the products of cell-free translation of polyadenylated RNA from spinach and pea. In both cases, the precursor is larger than the mature protein by 4000-5000 daltons. Upon incubation of post-ribosomal supernatants of the in vitro protein synthesis mixtures with purified intact chloroplasts, the pea and spinach precursors are transported interchangeably into the chloroplasts and processed to the mature size and charge. Moreover, the newly transported small subunits are found to assemble with endogenous large subunits to form the holoenzyme. In contrast, a precursor to the Chlamydomonas reinhardtii small subunit is not taken up by higher plant chloroplasts, indicating the specificity of the transport events. Together, these results demonstrate that the in vitro reconstruction of the post-translational transport of the higher plant precursors is physiologically significant.