Iliofemoral technique modification using an anchor screw as treatment of canine traumatic hip luxation - case report / [Modificação da técnica de sutura iliofemoral com uso de parafuso âncora no tratamento da luxação coxofemoral traumática em cães - relato de caso]

Twelve dogs with traumatic hip luxation were selected for surgical intervention with a modified iliofemoral suture technique using an anchor screw to substitute the passage of suture material through a perforated tunnel in the ilium. Six procedures were performed with non-absorbable suture and other six with absorbable suture materials. These cases were evaluated at 15, 30, 60, and 90 days after surgery by performing an ambulation analysis and palpation of the joint. In all cases, there was a return of partial and total limb support in an average of 3 and 19 postoperative, respectively. The fixation strategy of the suture material in the ilium using an anchor screw proved to be efficient with a smaller surgical approach and lesser surgical difficulty, maintaining joint congruence in acute as chronic luxation cases. The use of absorbable and non-absorbable sutures had excellent clinical results, but there was a subjective superiority of the first ones, once 4 dogs of the non-absorbable group presented some discomfort during the postoperative palpation of the joint, 90 days after surgery.(AU)

Doze cães com luxação coxofemoral traumática foram submetidos à intervenção cirúrgica de sutura iliofemoral modificada com uso de parafuso âncora substituindo a passagem de fio através de túnel perfurado no ílio. Seis procedimentos foram realizados com fio não absorvível, e outros seis com fio absorvível. Os casos foram avaliados aos 15, 30, 60 e 90 dias após a cirurgia, por meio de análise de deambulação e palpação articular. Em todos os casos, houve retorno de suporte parcial e total do peso no membro operado, em média, aos três e 19 dias de pós-operatório, respectivamente. A estratégia de fixação do fio de sutura no ílio com parafuso âncora se mostrou eficaz, permitindo uma abordagem cirúrgica menos invasiva, com menor dificuldade na execução, garantindo manutenção da congruência articular tanto em quadros de luxação aguda como crônica. O uso de fio absorvível e não absorvível teve bons resultados clínicos, porém houve uma superioridade subjetiva do primeiro, uma vez que quatro pacientes do grupo fio inabsorvível mostraram desconforto à palpação da articulação aos 90 dias após a cirurgia.(AU)

Long-term assessment of a modified tibial tuberosity advancement technique in dogs / Avaliação em longo prazo da técnica de avanço da tuberosidade tibial modificada em cães

The purpose of study was to assess long-term clinical and radiographic aspects of dogs' stifle joints which had undergone a modified tibial tuberosity advancement technique (mTTA). A total of 15 stifles that had undergone mTTA for CCL disease of 11 patients were included in this study. Assessments involved patient's gait analysis, cranial drawer and tibial compression tests, stifle goniometry range of articular motion, thigh and leg girth and radiographic evidence of progression of osteoarthrosis. Variables were compared between operated and healthy limbs and among moments (M0) on the early postop; (M1) 120 days postop; and (M2) approximately 5 years following surgery. A questionnaire regarding owner's perceptions after approximately 5 years of surgery was assessed. Most dogs presented positive response to cranial drawer and tibial compression tests on operated knees. There was also decrease on goniometry and thigh girth and increase in leg girth. Radiographic evidence of progression of osteoarthritis was seen especially on the long-term follow-up (M2). On gait analysis, most animals presented some degree of lameness in different conditions, in contrast to owners' perceptions. Osteoarthritis still develops in dogs following mTTA surgery for CCL disease. However, owners were overall satisfied with their recovery and would be willing to accept indication of mTTA for dogs with ruptured CCL.(AU)

O objetivo deste estudo foi avaliar, em longo prazo, aspectos clínicos e radiográficos do joelho de cães submetidos à técnica modificada de avanço da tuberosidade da tíbia (mTTA). Um total de 15 joelhos de 11 pacientes foram submetidos à mTTA para correção de doença do ligamento cruzado cranial. A avaliação envolvia análise de marcha do paciente, teste de compressão tibial e de gaveta, goniometria do joelho para amplitude articular, circunferência das pernas e coxas e evidência radiográfica de progressão da osteoartrose. As variáveis foram comparadas entre membros operados e saudáveis e entre os momentos (M0) no pós-operatório imediato; (M1) 120 dias de pós-operatório; e (M2) aproximadamente cinco anos após a cirurgia. Foi avaliado um questionário sobre as percepções do proprietário após aproximadamente cinco anos de cirurgia. A maioria dos cães apresentou resposta positiva aos testes de gavetas e de compressão tibial em joelhos operados. Houve também diminuição na goniometria e na circunferência da coxa e aumento do perímetro das pernas. Evidências radiográficas de progressão da osteoartrite foram observadas especialmente no seguimento de longo prazo (M2). Na análise de marcha, a maioria dos animais apresentou algum grau de claudicação em diferentes condições, em contraste com as percepções dos proprietários. A osteoartrite ainda se desenvolve em cães após a cirurgia de mTTA para doença CCL. No entanto, os proprietários estavam, em geral, satisfeitos com a recuperação dos animais e estavam dispostos a aceitar a indicação de mTTA para cães com doença do ligamento cruzado cranial.(AU)

Osteossíntese minimamente invasiva com placa bloqueada (Mipo) sem a utilização de intensificadores de imagem nas fraturas de tíbia em cães / Minimally invasive osteosynthesis with locked plate with no intraoperative imaging for tibial fractures in dogs

Avanços na compreensão da biologia óssea e das complicações das fraturas têm levado a modificações na abordagem da fixação interna, sendo a osteossíntese minimamente invasiva com placa (Mipo) uma das recentes evoluções para o manejo biológico das fraturas. O objetivo do presente estudo foi avaliar o uso de placas bloqueadas aplicadas de forma minimante invasivas em fraturas da tíbia de cães, sem a utilização de intensificadores de imagem intraoperatória. Foram utilizados 10 cães que apresentavam fraturas da tíbia, de idades e raças variadas. Os animais foram avaliados quanto à deambulação, à intensidade do edema, e exames radiográficos foram realizados nos períodos zero, 15, 30, 60, 90 e 120 dias de pós-operatório. Após 15 dias de pós-operatório, 80% dos cães já utilizavam o membro ao caminhar e apresentavam formação de edema discreto a ausente no membro operado. Após 30 dias de pós-operatório, todos os cães já apoiavam o membro ao caminhar e não apresentavam formação de edema. Todos os pacientes apresentaram sinais radiográficos de início de consolidação óssea após 15 dias de pós-operatório e pôde-se observar que a união clínica ocorreu em média aos 42,5 dias. O tratamento de fraturas de tíbia por meio da Mipo é alternativa eficaz, resultando em utilização precoce do membro e união clínica efetiva, e pode ser realizado sem uso de intensificadores de imagem.(AU)

Advances in the comprehension of bone healing and complications of treatment of fractures have occurred. Less invasive procedures have been advocated. A minimally invasive plate osteosynthesis (MIPO) is one of the ways to avoid tissue damage. The aim of this study was to evaluate the use of MIPO in tibial fractures in dogs, with no intraoperative imaging. Ten dogs presenting tibial fracture were used. Deambulation, swelling and radiographic changes were evaluated preoperatively, just after the procedure and at 15, 30, 60, 90 e 120 days postoperatively. After 15 days of surgery 80% dogs were able to walk with slight swelling or without swelling. After 30 days all dogs have supported the member to walk and had no swelling. All dogs presented initial radiographic signs of bone healing after 15 postoperative days and it can be seen that the average clinical union occurred at 42.5 days. The treatment of tibial fractures by MIPO is an effective alternative resulting in early and effective clinical union and can be performed without the use of image intensifiers.(AU)

Brain neuroimaging of domestic cats: correlation between computed tomography and cross-sectional anatomy / Neuroimagem cerebral de gatos domésticos: correlação entre a tomografia computadorizada e a anatomia seccional

Computed tomography of the brain is necessary as part of the diagnosis of lesions of the central nervous system. In this study we used six domestic cats, male or female, aged between one and five years, evaluated by Computed Tomography (CT) examination without clinical signs of central nervous system disorders. Two euthanized animals stating a condition unrelated to the nervous system were incorporated into this study. The proposal consisted in establishing detailed anatomical description of tomographic images of normal brain of cats, using as reference anatomical images of cross sections of the stained brain and cranial part, with thicknesses similar to the planes of the CT images. CT examinations were performed with and without intravenous iodinated contrast media for live animals. With one euthanized animal, the brain was removed and immediately preserved in 10% formalin for later achievement in cross-sectional thickness of approximately 4mm and staining technique of Barnard, and Robert Brown. The head of another animal was disarticulated in the Atlanto-occipital region and frozen at -20ºC then sliced to a thickness of about 5mm. The description of visualized anatomical structures using tomography is useful as a guide and allows transcribing with relative accuracy the brain region affected by an injury, and thus correlating it with the clinical symptoms of the patient, providing additional information and consequent improvement to veterinarians during the course of surgical clinic in this species.(AU)

A tomografia computadorizada do cérebro é de suma importância como parte do diagnóstico de lesões do sistema nervoso central. Neste estudo, foram utilizados seis felinos domésticos, machos ou fêmeas, com idade entre um e cinco anos, avaliados pela tomografia computadorizada (TC), sem sinais clínicos de distúrbios do sistema nervoso central. Dois cadáveres indicando a condição relacionada com o sistema nervoso foram incorporados ao presente estudo. A proposta consistiu na criação da descrição anatômica detalhada das imagens tomográficas de cérebro normal de gatos, usando como referência a imagens anatômicas de secções transversais do cérebro coradas, realizadas com espessuras similares aos planos das imagens tomográficas. Exames de TC foram realizados com e sem contraste iodado intravenoso para os animais vivos. Em um dos cadáveres, o cérebro foi removido e imediatamente preservado em formalina a 10% para a realização posterior da técnica de coloração de Barnard e Robert Brown, além de cortes transversais com espessura de, aproximadamente, 4mm. O crânio do outro cadáver foi desarticulado na região atlanto-occipital e congelado a -20ºC. Em seguida, foi realizado corte com espessura de cerca de 5mm. A descrição das estruturas anatômicas visualizadas foi útil e serve como guia, permitindo descrever com relativa precisão a estrutura cerebral normal ou lesionada, e, portanto, correlacionando-a com os sintomas clínicos de um paciente, o que fornece informações adicionais e consequente aprimoramento de veterinários no curso de clínica cirúrgica para essa espécie.(AU)

Detection of Leptospira spp. in wild Phrynops geoffroanus (Geoffroy's side-necked turtle) in urban environment.

Leptospira spp., a zoonotic agent relevant for public health, occurs frequently in tropical regions. The aquatic environment represents a viable survival and transmission pathway. This study aimed to investigate the presence of anti-Leptospira spp. antibodies in Phrynops geoffroanus (Geoffroy's side-necked turtle) serum samples using the microagglutination test (MAT), and Leptospira spp. in gastric and cloacal lavage samples using the polymerase chain reaction (PCR) technique. Antibodies against nine different Leptospira spp. serovars were detected in 45.45% (30/66) of the serum samples. Specific amplification of Leptospira spp. genomic material (331bp) was observed in 16.67% (11/66) of the samples. In conclusion, these freshwater testudines host Leptospira spp. and eliminate them. This situation may represent a risk to public health, especially to people who use urban streams for fishing and recreational activities. Additionally, we described some Leptospira spp. serovars, not yet reported in testudines, detected here in P. geoffroanus.

Follistatin concentrations in follicular fluid of normal and polycystic ovaries.

Follistatin (FS) is an activin/inhibin binding protein which is believed to act in an autocrine/paracrine manner to regulate growth and differentiation. Although FS has been identified in human follicular fluid, it remains unclear how its concentration changes during selection and atresia, and what the concentrations of FS are in follicles of women with polycystic ovary syndrome (PCOS). Towards this goal, we have measured by radioimmunoassay the concentrations of FS in follicular fluid obtained from dominant and atretic cohort follicles of normal cycling women, preovulatory follicles of in-vitro fertilization (IVF) patients, and small Graafian follicles of patients with PCOS. In all cases, the follicular fluid concentration of FS was much higher (approximately 100-fold) than that reported in serum. The FS concentrations (ng/ml) were 203 +/- 42 (normal dominant), 185 +/- 17 (atretic cohort), 185 +/- 5 (IVF), and 250 +/- 14 (PCOS). There was no statistical difference between these mean values of FS. Further, there were no significant correlations between the follicular fluid concentrations of FS and the concentrations of oestradiol, progesterone, or androstenedione. These results indicate that human Graafian follicles, regardless of whether they are healthy or atretic, normal or PCOS, contain high steady-state concentrations of FS in the micro-environment. Collectively, these data fit with the hypothesis that major increases and decreases in the concentration of FS in the micro-environment may not play a key role in the mechanisms of selection, atresia, and PCOS in women. The possibility of regulation of intrinsic activin and inhibin activity through FS binding is discussed.

Endodermal sinus tumor arising from a dysgenetic gonad in a 46,XX female combined with müllerian agenesis.

The occurrence of neoplastic transformation in a dysgenetic gonad of a female with 46,XX karyotype is very rare. Moreover, to our knowledge, a case of endodermal sinus tumor arising from that setting combined with Müllerian agenesis has not been reported. This case suggests the possibility of development of cancer in a dysgenetic ovary of a female even with 46,XX or Müllerian agenesis.

SPECT for differential diagnosis of dementia and correlation of rCBF with cognitive impairment.

99mTc-HM-PAO single photon emission computed tomography (SPECT) was used to image 30 patients referred for the assessment of dementia. SPECT images revealed various patterns of regional cerebral perfusion (rCBF) in the subgroups of patients with the clinical diagnoses of Alzheimer's disease (AD, n = 14), Pick's disease (n = 1), and multi-infarct dementia (n = 7). In three patients, SPECT clarified the clinical differential diagnostic possibilities. Using a relative rCBF quantification technique, the relationship between specific cognitive impairments and rCBF in the AD patients was determined. There was a significant correlation between language impairment and left hemisphere hypoperfusion, whereas, apraxia correlated with hypoperfusion in the left parietal region. Thus, HM-PAO SPECT is useful as an aid in the differential diagnosis of dementia and the technique of relative rCBF quantification with SPECT may contribute to the understanding of the clinico-anatomical relations of cognitive deficits in dementia.

Technetium-99m-HMPAO SPECT, CT and MRI in the evaluation of patients with chronic traumatic brain injury: a correlation with neuropsychological performance.

UNLABELLED: The purposes of this study were: (1) to compare 99mTc-hexamethylpropyleneamineoxime (HMPAO) SPECT with CT and MRI in chronic traumatic brain injury (TBI) patients and (2) to correlate both functional and structural neuroimaging measurements of brain damage with neuropsychological (NP) performance. METHODS: Twenty-nine patients (minor TBI, n = 15 and major TBI, n = 14) and 17 normal controls (NC) underwent HMPAO SPECT, CT, MRI and NP testing. Imaging data were analyzed both visually and quantitatively. RESULTS: Nineteen (66%) patients showed 42 abnormalities on SPECT images, whereas 13 (45%) and 10 (34%) patients showed 29 abnormalities on MRI and 24 abnormalities on CT. SPECT detected relatively more abnormalities than CT or MRI in the minor TBI subgroup. The TBI group showed impairment on 11 tests for memory, attention and executive function. Of these, the anterior-posterior ratio (APR) correlated with six tests, whereas the ventricle-to-brain ratio (VBR), a known structural index of a poor NP outcome, correlated with only two tests. CONCLUSION: In evaluating chronic TBI patients, HMPAO SPECT, as a complement to CT or MRI, may play a useful role by demonstrating brain dysfunction in morphologically intact brain regions and providing objective evidence for some of the impaired NP performance.

Neuroanatomical localization for clinical SPECT perfusion brain imaging: a practical proportional grid method.

For the purpose of facilitating anatomical localization in interpretation of 99Tcm-hexamethylpropyleneamine oxime (HMPAO) brain single photon emission tomographic (SPECT) scans, a stereotaxic proportional grid system was applied in the form of an interactive computer program. This method takes advantage of a rotating gamma camera system which permits planar scout imaging for the determination of anatomical reference lines, and standardization of tomographic slices for brain size. Using measurements made on a lateral planar HMPAO image, proportional grids were constructed onto standardized transaxial images. This method was implemented for 33 clinical HMPAO SPECT studies. It required less than 15 min of an operator's time. This simple and practical neuroanatomical localization technique can be instrumental as an aid to the interpretation of routine clinical HMPAO SPECT images.

Assessment of regional cerebral perfusion by 99Tcm-HMPAO SPECT in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a severely disabling illness of uncertain aetiology. It is characterized by a chronic, sustained or fluctuating sense of debilitating fatigue without any other known underlying medical conditions. It is also associated with both somatic and neuropsychological symptoms. Both physical and laboratory findings are usually unremarkable. Regional cerebral blood flow (rCBF) was assessed in 60 clinically defined CFS patients and 14 normal control (NC) subjects using 99Tcm-hexamethylpropyleneamine oxime (99Tcm-HMPAO) single photon emission computed tomography (SPECT). Compared with the NC group, the CFS group showed significantly lower cortical/cerebellar rCBF ratios, throughout multiple brain regions (P < 0.05). Forty-eight CFS subjects (80%) showed at least one or more rCBF ratios significantly less than normal values. The major cerebral regions involved were frontal (38 cases, 63%), temporal (21 cases, 35%), parietal (32 cases, 53%) and occipital lobes (23 cases, 38%). The rCBF ratios of basal ganglia (24 cases, 40%) were also reduced. 99Tcm-HMPAO brain SPECT provided objective evidence for functional impairment of the brain in the majority of the CFS subjects. The findings may not be diagnostic of CFS but 99Tcm-HMPAO SPECT may play an important role in clarifying the pathoaetiology of CFS. Further studies are warranted.

Technetium-99m-HMPAO SPECT in the evaluation of patients with a remote history of traumatic brain injury: a comparison with x-ray computed tomography.

The functional imaging modality has potential for demonstrating parenchymal abnormalities not detectable by traditional morphological imaging. Fifty-three patients with a remote history of traumatic brain injury (TBI) were studied with SPECT using 99mTc-hexamethylpropyleneamineoxime (HMPAO) and x-ray computed tomography (CT). Overall, 42 patients (80%) showed regional cerebral blood flow (rCBF) deficits by HMPAO SPECT, whereas 29 patients (55%) showed morphological abnormalities by CT. Out of 20 patients with minor head injury, 12 patients (60%) showed rCBF deficits and 5 patients (25%) showed CT abnormalities. Of 33 patients with major head injury, 30 patients (90%) showed rCBF deficits and 24 patients (72%) showed CT abnormalities. Thus, HMPAO SPECT was more sensitive than CT in detecting abnormalities in patients with a history of TBI, particularly in the minor head injury group. In the major head injury group, three patients showed localized cortical atrophy by CT and normal rCBF by HMPAO SPECT. In the evaluation of TBI patients, HMPAO SPECT is a useful technique to demonstrate regional brain dysfunction in the presence of morphological integrity as assessed by CT.

Cloning, sequencing and overexpression of the gene for prokaryotic factor EF-P involved in peptide bond synthesis.

A soluble protein EF-P (elongation factor P) from Escherichia coli has been purified and shown to stimulate efficient translation and peptide-bond synthesis on native or reconstituted 70S ribosomes in vitro. Based on the partial amino acid sequence of EF-P, 18- and 24-nucleotide DNA probes were synthesized and used to screen lambda phage clones from the Kohara Gene Bank. The entire EF-P gene was detected on lambda clone #650 which contains sequences from the 94 minute region of the E.coli genome. Two DNA fragments, 3.0 and 0.78 kilobases in length encompassing the gene, were isolated and cloned into pUC18 and pUC19. Partially purified extracts from cells transformed with these plasmids overrepresented a protein which co-migrates with EF-P upon SDS polyacrylamide gel electrophoresis, and also exhibited increased EF-P mediated peptide-bond synthetic activity. Based on DNA sequence analysis of this gene, the EF-P protein consists of 187 amino acids with a calculated molecular weight of 20,447. The sequence and chromosomal location of EF-P establishes it as a unique gene product.

A proposed role for IF-3 and EF-T in maintaining the specificity of prokaryotic initiation complex formation.

Initiation factor-free 30S subunits of E. coli ribosomes bind aminoacyl-tRNAs more efficiently than fMet-tRNA(fMet). Elongator-tRNA binding was unaffected by IF-1 or IF-2 but was inhibited by IF-3. Their combination reduced this binding up to 40% and stimulated that of fMet-tRNA(fMet). Unexpectedly, EF-T also prevented elongator-tRNA binding by complexing both to the 30S and to the aminoacyl-tRNAs. Using AUGU3 as mRNA, elongator-tRNAs competed with fMet-fRNA(fMet) and with tRNA(fMet), fMet-tRNA(fMet) reacted with puromycin after addition of 50S subunits suggesting that it occupied the P site. EF-T directed binding of phe-tRNA to the 30S.AUGU3 complex at the A site only if fMet-tRNA(fMet) or tRNA(fMet) filled the P/E site. We propose that one function of EF-T may be to prevent the entry of aminoacyl-tRNAs into the 30S particle during initiation. The possibility that a special site for fMet-tRNA resides on 16S rRNA is also discussed.

Regulation of gene expression at the translational level. The rescue factor reverses thermosensitive protein synthesis in N4316, a conditionally-lethal mutant of Escherichia coli defective in translation.

Extracts of the conditionally-lethal mutant Escherichia coli N4316 are defective in a newly described translation factor, the rescue protein. We have analyzed the in vitro translation products of this mutant by gel electrophoresis during normal and arrested synthesis at the permissive and non-permissive temperatures. Translation programmed with MS2 bacteriophage RNA at the non-permissive temperature results in highly reduced synthesis of the coat protein with no detectable levels of the maturation and replicase products. Thus the relative number of copies of proteins synthesized by the ribosomes is altered in this mutant. In addition, there is mistranslation of the coat gene which results in the overproduction of the phage encoded no. 7 protein. Aberrant synthesis is also reflected in the increased read-through of termination codons during synthesis directed by phage RNAs harbouring amber mutations in the coat cistron. The rescue protein, purified from the parental strain, is able to complement the thermosensitive defect and restore proper synthesis. Biochemical characterization of the defect in the absence of rescue shows no detectable deficiency in the extent of initiation complex formation in reactions inhibited with sparsomycin. Peptidyltransferase is fully active as judged by the kinetics of formylmethionine-puromycin formation. However, rescue does exert an effect at the level of termination. In addition, the thermolability of the mutant can be reversed by dissociating 70S ribosomes into 30S and 50S subunits. Based on these and other observations, we propose tht rescue mediates a novel function in the association/dissociation of ribosomal subunits which is essential to the accuracy and efficiency of translation.

Recognition of translational termination signals.

Ribosomes can specifically shift at certain codons so that the mRNA is read in two different reading frames. To determine if frameshifting occurs at the level of termination, polymers of defined sequence containing AUG, a coding sequence and an in- or out-of-phase nonsense codon were used to bind a termination substrate or to program synthesis and release of dipeptides in a highly purified in vitro translation system. fMet-tRNA bound to ribosomes with AUGUAA, AUGUAAn, AUGUUU, AUGUUA or AUGUAn was not a substrate for release factor RF-1. In contrast, AUGU1UAA, AUGU3UAAn, AUGU4UAAn, AUGU5UAAn effected RF-1-dependent release of fMet from ribosomes. This suggests that nonsense codons can stimulate release whether they occur in- or out-of-phase. Addition of exogenous UAA and RF-1 stimulated release with all oligonucleotides tested. Propagation restricted the RF-1-dependent recognition of out-of-phase nonsense codons but did not restrict recognition of in-phase UAA in AUGU3UAAn. Release of dipeptides from ribosomes programmed with AUGU4UAAn occurred without EF-G and with a mutant lacking EF-G activity, suggesting that out-of-phase termination can occur prior to translocation outside the ribosomal A-site. We propose that the ribosome X RF complex is required to complete proteins, but is also able to frameshift at a nonsense codon resulting in occasional out-of-phase termination of protein synthesis.

Reconstruction of peptidyltransferase activity on 50S and 70S ribosomal particles by peptide fragments of protein L16.

Ribosomal protein L16 was digested with Staphylococcus aureus protease V8 and the resulting peptides were separated by reversed-phase high-performance liquid chromatography. One of the fragments, identified by sequence analysis as the N-terminal peptide of L16, was shown to exhibit partial peptide-bond-formation and transesterification activities of peptidyltransferase upon reconstitution with L16-depleted 50S core particles. However, several proteins enhanced these activities. L15 increased both reactions when added to the reconstitution mixture, suggesting a limited capacity of the L16 peptide to incorporate into 50S core particles. In contrast, the interaction of L11 with the N-terminal peptide stimulated the transesterification reaction but not the peptide-bond-forming activity of ribosomes, indicating a different topological domain for these reactions. Also, EF-P, a soluble protein which reconstructs the peptide-bond formation and transesterification reactions on 70S ribosomes, stimulated both peptidyltransferase activities exhibited by the L16 N-terminal peptide.

Internal architecture of the core nucleosome: fluorescence energy transfer studies at methionine-84 of histone H4.

Chicken histone H4, labeled separately at Met-84 with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, was reassociated with unlabeled histones H2A, H2B, and H3 and 146 base pairs of DNA to produce fluorescently labeled nucleosomes having physical characteristics virtually the same as those of native core particles. Four types of particles were prepared containing respectively unlabeled H4, dansylated H4, fluoresceinated H4, and a mixture of the two labeled H4 molecules. Quantitative singlet-singlet energy-transfer measurements were carried out to determine changes in the distance between the two Met-84 H4 sites within the same nucleosome following conformational transitions which we have reported earlier. In the ionic strength range 0.1-100 mM NaCl, the distance between these sites is less than 2 nm except at 1 mM. Between 100 and 600 mM monovalent salt the distance separating the donor and acceptor fluors at Met-84 H4 increases to 3.8 nm. The conformational change centered around 200 mM NaCl is cooperative. Our results and those of others indicate that there is little unfolding of the histone octamer, at least around Met-84 H4, in the entire ionic strength range studied. A mechanism involving the rotation of the globular portion of H4 is proposed to account for this transition which occurs at physiological ionic strengths.

Intermolecular histone H4 interactions in core nucleosomes.

Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the pyrene-labeled nucleosome was characteristic of excited dimer (excimer) fluorescence, indicating that the single pyrene groups on the two H4 molecules are in close proximity in the reconstituted particle. Histone H4 was also labeled randomly at lysines with a group that contains two pyrene moieties separated by 12 A at most. Incorporation of this histone into nucleosome-like particles provides an excimer standard which does not depend on intermolecular interactions. The properties of the pyrene-containing nucleosome were examined as a function of ionic strength. It was found that the H4-H4 pyrene excimer fluorescence exhibited a cooperative disruption centered at 0.1 M NaCl which preceded increases in accessibility and environment polarity revealed by other fluors attached at the same site.

Conformations of the core nucleosome: effects of ionic strength and high mobility group protein 14 and 17 binding on the fluorescence emission and polarization of dansylated methionine-84 of histone H4.

Chicken histone H4 labeled at Met-84 with the fluor N-[(acetylamino)ethyl]-8-naphthyl-amine-1-sulfonic acid has been incorporated into a nucleosome which has physical characteristics virtually identical with those of native core nucleosomes. The fluorescence emission and polarization properties of the labeled nucleosome were measured as a function of ionic strength and the binding of high mobility group (HMG) proteins 14 and 17. Also, the accessibility of the fluor to the quenching agent acrylamide was determined. It was found that the fluorescence emission changes in the range 0.1-1000 mM NaCl are rather small and indicate that no major unfolding of the octamer structure occurs around Met-84 on H4 at least. Five or perhaps six discrete states were found in that ionic strength range. Each has a different accessibility to the quenching agent. The range of accessibilities varied from 9 X 10(-7) to 32 X 10(-7) mol-1 s-1 for 0.1-1000 mM NaCl, respectively. Polarization measurements showed that there was little change in the rotational relaxation lifetime of the fluor at ionic strengths less than 50 mM NaCl. Above this value, the rotational relaxation lifetimes decreased from 107 to 25 ns at 600 mM NaCl, indicating a moderately increased rotational freedom for the fluor. It is suggested that the histone octamer changes its degree of compaction in the range 0.1-600 mM NaCl but that no major protein unfolding occurs.(ABSTRACT TRUNCATED AT 250 WORDS)