Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation.

The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.

Expression of the calcium-sensing receptor on human antral gastrin cells in culture.

The presence of the extracellular calcium-sensing receptor on human antral gastrin cells was investigated. Reverse transcription PCR using mRNA isolated from gastrin cell- enriched cell cultures identified a product with a sequence identical to part of the human parathyroid-secreting cell calcium-sensing receptor. Immunocytochemistry with an antibody to the extracellular region of the receptor immunostained all gastrin cells (but not mucin or somatostatin cells), and detected appropriate-sized bands in Western blots of whole cell lysates. Increasing extracellular calcium levels from 0.5 to 9 mM stimulated gastrin release in a concentration-dependent manner, with maximal release obtained at 7.2 mM. A known agonist of the calcium receptor, spermine also stimulated gastrin release. Microfluorimetry of identified gastrin cells demonstrated that increasing extracellular calcium resulted in an initial rapid rise in intracellular calcium followed by a plateau level that returned to basal levels immediately after removal of the elevated calcium. The traces were consistent with activation of a receptor-mediated mechanism rather than a concentration-dependent influx of calcium. In conclusion, these data indicate that G cells express the calcium-sensing receptor, and that activation of the receptor may explain the acid rebound phenomenon associated with calcium-containing antacid preparations.

Inhibition of tumor cell invasion by a highly conserved peptide sequence from the matrix metalloproteinase enzyme prosegment.

The metastasis associated 72-kDa type IV collagenase is secreted as a latent proenzyme which is converted to an active 62-kDa form by autoproteolytic removal of an amino terminal profragment. The region immediately upstream from the cleavage site contains a highly conserved peptide sequence, MRKPRCGNPDV, which is present in all known members of the matrix metalloproteinase family. Evidence implicates the cysteine residue of this sequence as critical for maintenance of the latent form through coordination with the catalytic zinc atom of the active site. A synthetic peptide, TMRKPRCGNPDVAN (peptide 74), encompassing this conserved sequence, has been shown to inhibit the activated form of the 72-kDa type IV collagenase in vitro. In the present study we examine the ability of this peptide inhibitor to modulate tumor cell invasiveness. Peptide 74 and the control peptide 78, which contains a single substitution of serine for the "critical" cysteine residue, were added at 30 microM concentrations to the upper compartment of the Boyden chamber in the chemoinvasion assay using HT1080 and A2058 human tumor cells. In this assay a layer of reconstituted basement membrane, Matrigel, is coated onto chemotaxis filters and acts as a barrier to the migration of cells in the Boyden chambers. Only cells with invasive capacity can cross the Matrigel barrier. Peptide 74 containing the cysteine residue inhibited the invasion of both the HT1080 and A2058 cells through the Matrigel barrier; control peptide 78 was not inhibitory. Both peptides were shown to be without cytotoxic action and did not inhibit chemotaxis or affect cell number. This study demonstrates that addition of an excess peptide containing the matrix metalloproteinase prosegment inhibitory sequence can inhibit invasive activity at the cellular level and suggests that this may be a useful strategy to modulate tumor cell invasiveness in vivo.

TIMP-2 expression modulates human melanoma cell adhesion and motility.

Invasion of cells across extracellular matrix barriers requires attachment of cells to the matrix, creation of a proteolytic defect in the matrix, and migration of the cells through the defect. To date, alterations in the balance between matrix metalloproteinases (MMPs) and their inhibitors have been shown to alter cellular invasion only through effects on matrix degradation. We used a retroviral infection system to over- and underproduce tissue inhibitor of metalloproteinase-2 (TIMP-2) in human A2058 melanoma cells. Our results indicate that altering the balance of MMPs and TIMP-2 through genetic manipulation of TIMP-2 production modulates not only proteolysis of the extracellular matrix but also cell attachment to the extracellular matrix and motility of cells through matrix components. Altering the production of TIMP-2 also results in the ability of cells to form foci. These results implicate the MMPs and their inhibitors in all aspects of the cellular invasion cascade. This supports the hypothesis that highly invasive cell lines establish a balance of MMPs and inhibitors that is optimal for invasion, and alteration of this balance in either direction results in perturbation of the invasive phenotype.

Acute tears of the anterior cruciate ligament. Surgical versus conservative treatment.

In a prospective eight-year study comparing surgical and conservative treatment of acute tears of the anterior cruciate ligament, ninety-two patients in whom a tear had been confirmed either by arthroscopy or by arthrotomy were treated and could be followed. The choice of treatment was determined entirely by whether the result of a pivot-shift test was graded as absent, trace, or mild (non-operative treatment) or as moderate or severe (surgical treatment). Surgical treatment consisted of primary repair of the torn ligament and augmentation with a patellar tendon graft. Twenty-two patients were treated non-operatively, and the results were evaluated after twenty-four to eighty-two months (average, forty-eight months). Seventy patients were treated surgically, and fifty-two of them (approximately 70 per cent) returned for follow-up after twenty-four to 100 months (average, forty-eight months); an additional eighteen patients answered a questionnaire. In the non-operative group, about half of the results were graded as excellent or good and half, as fair or a failure. In the surgical group, all but two of the patients had an excellent or a good result; two patients had a fair result. No result was graded as a failure. The results of this study suggest that when the pivot-shift test is not strongly positive, half of the patients will do reasonably well after treatment with a non-operative program of functional rehabilitation. The patients in this study who had a more unstable knee had far better results after a repair and augmentation procedure than have been previously reported after primary repair alone.

Erythrocyte survival following intraoperative autotransfusion in spinal surgery: an in vivo comparative study and 5-year update.

A 5-year prospective study of intraoperative blood transfusion in 239 patients, most of whom had major spinal surgery, has been completed. Autotransfusion is safe, practical, and it reduces donor blood requirements and total blood loss by 50%. It eliminates host versus graft reactions and disease transmission. In 33 patients having major spinal surgery, the in vivo survival of autologous, homologous, and processed red blood cells (RBC) using the Cell Saver System were compared. The RBC survival studies were performed using chromium51 isotope labeling technique. The long-term survival of processed RBCs was normal over a 30-day period. There was no significant difference in the survival rate of the three groups studied.

Semimembranosus tendinitis: an overlooked cause of medial knee pain.

A significant number of patients presenting with nontraumatic chronic medial knee pain have been found, after careful examination, to have chronic semimembranosus tendinitis. Of 115 patients diagnosed and treated for semimembranosus tendinitis over a 5 year period, 10 patients were refractory to conservative treatment. The purpose of this paper is to discuss the symptomatology and diagnosis of semi-membranous tendinitis, and to relate the treatment that these 10 patients underwent, i.e., surgical exploration, drilling of the insertion site, and semitendinosus tendon transfer. Most cases of semitendinosus tendinitis respond to conservative therapy; however, if surgical treatment is considered, arthroscopy should probably be performed at the time of surgery. In this way, it is possible to determine the presence of significant intraarticular abnormalities that may have a causal relationship with the onset of semimembranosus tendinitis.

Effects of electrical and electromagnetic stimulation after anterior cruciate ligament reconstruction.

A need exists to develop new methods of neuromuscular electrical stimulation (NMES) that are both effective and relatively pain-free. The purpose of this pilot study was to determine the effects of both NMES and a new method of electromagnetic (NMES/PEMF) stimulation for reducing girth loss and for reducing pain and muscle weakness of the knee extensor muscles in patients during the first 6 weeks after reconstructive surgery of the anterior cruciate ligament (ACL). Seventeen patients receiving ACL reconstructive surgery participated as a control group (N = 3), as an NMES group (N = 7), and with combined NMES and magnetic field stimulation (NMES/PEMF) (N = 7). Patients receiving NMES/PEMF rated each type of stimulation for perceived pain and were measured for their torque. Torque results revealed a mean decrease of 13.1% for NMES/PEMF patients. The mean percent of thigh girth decreased 8.3% for controls, 0.5% for NMES, and 2.3% for NMES/PEMF patients. The NMES/PEMF patients rated NMES as causing about twice the pain intensity as NMES/PEMF during treatments. As a result of this data, the authors conclude that both NMES and NMES/PEMF are effective in reducing girth loss and that NMES/PEMF is less painful than NMES alone in treating patients after ACL reconstruction.

A proposed natural history of symptomatic anterior cruciate ligament injuries of the knee.

This article highlights the symptoms and clinical findings associated with anterior cruciate ligament instability. The symptomatic anterior cruciate ligament-deficient knee presents as an acute isolated incident or as a chronic progression of recurrent episodes of instability. The results of 71 patients diagnosed with an anterior cruciate ligament instability to the knee are reported. No distinction was made between the acute and chronic injuries as both patient populations received the same surgical procedure. The Lachman's test and the pivot shift test are the clinical examinations used to document cruciate ligament instability. The recommended procedure of choice for the symptomatic anterior cruciate-deficient knee is an autogenous patella tendon graft, bone-tendon-bone preparation, arthroscopically placed in an isometric position within the knee joint. A rehabilitation program for the reconstructed knee is outlined.

Enhanced wet air oxidation of sediment contaminated with PCBs.

Wet air oxidation (WAO) is investigated as a method of treating river sediments contaminated with polychlorinated biphenyls (PCBs). Aqueous slurries containing 2.5% (w/w) sediment were oxidized with oxygen in a one liter, high-pressure, batch reactor at temperatures up to 250 degrees C. Concentrations of PCBs adsorbed on the sediment and reactor surfaces and dissolved in the water and gas phases after oxidation were determined by high-resolution gas chromatography. Results indicate that no significant wet oxidation of PCBs in sediment slurries occurs for temperatures at or below 250 degrees C. However, during reactor heat-up, significant degradation of PCBs occurred at high temperature regions near the reactor wall even when bulk fluid temperature was quite low. A variety of amendments were tested to determine their effect on PCB oxidation. These amendments included hydrogen peroxide, a readily degraded organic compound (phenol), and homogeneous copper catalyst. Only hydrogen peroxide addition resulted in a significant degradation of PCBs. The addition of phenol did not result in enhanced degradation of PCBs through kinetic coupling as has been observed for other recalcitrant organic compounds.

Post-arthroscopic pulmonary edema in two healthy teenage athletes.

Outpatient arthroscopic knee surgery carries with it certain risk factors similar to those accompanying other major knee procedures. These risks may be related to the complexity of the procedure, exposure to anesthesia, or risk factors from previous medical history. Two cases of acute pulmonary edema following arthroscopic knee surgery in otherwise healthy teenage athletes are presented. Both patients developed acute respiratory distress in the recovery room after uneventful arthroscopic knee surgery. The patients in both cases recovered and were able to return to sporting activity with no sequelae. The similarities in both cases prompted a retrospective investigation of the events from the induction of general anesthesia to the admission of the patients to the intensive care unit. Several possible causes of acute post-operative pulmonary edema include fluid overload, cardiac arrhythmia, respiratory depression, systemic drug reaction and sickle cell trait or disease. Outpatient arthroscopy still remains the procedure of choice for meniscal pathology of the knee, but the surgeon and the anesthesia personnel must be aware of and prepared for pulmonary complications that may arise in the immediate post-operative period.

Results of the Kentucky high school football knee injury survey.

The study attempted to: 1) determine knee injury and reinjury incidence of Kentucky high school football players; 2) relate results to initial care provider, treatment following initial physician exam, time lost from injury, injury type, player position, and team size; and 3) assess coaches opinions and practices about lateral prophylactic knee brace (LPKB) usage and effectiveness. A post season, mail-in coaches' survey (50.2% return, 101/201) collected these data. Returned surveys represented 4690 players with average team size (x +/- SD) of 43 +/- 13. Two hundred fifty seven reported knee injuries yielded .055 knee injuries/player with .04 knee injuries/player being "new" and .015 knee injuries/player recurring (27% of reported knee injuries) during the season. Games accounted for 56.4% (56/101) of reported knee injuries. Coaches generally believed that LPKB usage prevented knee injuries (56.4%, 56/101) and allowed LPKB usage (92.1%, 93/101), however only 8.3% (8/101) required their wear (interior linemen 50%, linebackers 25%, entire team (25%). Interior linemen had the greatest number of knee injuries, followed by offensive backs and linebackers. Most knee injuries (81%, 208/257) were out 3-6 weeks or less, 64% (164/257) involved sprains or contusions, 38% (97/257) were treated surgically (alone or with rehabilitation) and 36% (92/257) were treated solely with rehabilitation. Total knee injury and reinjury incidence were under-estimated compared to existing reports. Improved injury recording methods, and post-symposia coaches evaluation are recommended.

The efficacy of optical and pharmacological penalization.

PURPOSE: Optical and pharmacological penalization of sound eyes are infrequently used alternatives to occlusion for treating amblyopia. The authors evaluated the efficacy of penalization as their primary treatment of amblyopia. METHODS: One hundred sixty-six patients underwent penalization treatment for strabismic or anisometropic amblyopia for a minimum of 3 months. Both atropine and optical penalization methods were used. RESULTS: Visual acuity improved in 67 (77%) of 87 patients treated with optical penalization. There was a significant improvement of the geometric mean visual acuity of the amblyopic eyes from 20/38 to 20/28 (P < 0.001). Visual acuity of 60 (76%) of 79 patients treated with pharmacological penalization improved. There was a significant improvement of mean visual acuity of the amblyopic eyes from 20/61 to 20/40 (P < 0.001). Neither therapy produced an instance of occlusion amblyopia. Thirteen patients discontinued therapy because of blur or discomfort. CONCLUSION: This study demonstrates that penalization methods are effective methods for the treatment of amblyopia, with a low risk of occlusion amblyopia. Patient acceptance of these methods was excellent. Penalization should be considered more often for the primary treatment of amblyopia.

Structured review of enamel erosion literature (1980-1998): a critical appraisal of experimental, clinical and review publications.

OBJECTIVE: To attain an objective account of the methods to measure enamel erosion used in 1980-1998 publications, a structured review of the literature was undertaken. METHODS: Inclusion and exclusion criteria were applied to 731 clinical/experimental research and review reports. Eighty-five included papers were subsequently rated according to 'hierarchy of evidence' guidelines to assess the strength of the report's design and the relevance of the evidence to replicating enamel erosion in vivo in humans. Scores were assigned to rate each aspect in the guidelines. RESULTS: A total of 16 clinical, 13 review and 56 experimental papers were assessed; 36.4% were published during 1996-1998. Excluding reviews, 16 papers were qualitative and 56 quantitative; 51 used human enamel. Our classification yielded nine groups of methods (five scoring systems and 26 measurement techniques). CTFPHE (Can Med Assoc J 1992; 147: 443) grading of research reports indicated that 2.8% provided evidence grade I; 20.8%, grade IIa; 63.9%, grade III; and 12.5%, grade IV. CONCLUSIONS: There has been a consistent increase in the body of knowledge. The overall quality of publications has not substantially changed over time. Experimental studies were more often quantitative, and quantitative studies had better research designs. No single group of research methods had obviously superior research designs.

Incidence, mechanism of injury, and treatment of fractures of the patella in children.

Fractures of the patella in skeletally immature patients are rare. The charts of 185 patients treated for patella fractures at the University of Kentucky Medical Center between 1976 and 1988 were retrospectively reviewed. The 12 patients of these 185 aged 8 to 16 years were included in this study. The incidence was calculated to be 6.5% of all patella fractures. All patients studied were male with an average age of 12.7 years. Sleeve fractures were the most common type of patella fracture observed (five), followed by transverse fractures (four). Ten of the 12 cases required operative management ranging from irrigation and debridement to open reduction and internal fixation. Partial patellectomy was performed when indicated. Indications for operative management in this age group were similar to those for adults. As in adults, the mechanism of injury was predominantly motor vehicle and motorcycle crashes. Laws requiring seatbelt restraints for children should have a positive effect on the incidence of such fractures resulting from dashboard injuries. One mechanism of injury not reported previously was that of a flexed knee striking the gym wall after performing a basketball lay-up because the basket was placed flush with the wall.

Male drinking and drunkenness in Middletown.

Based on anthropological evidence, MacAndrews and Edgerton state that drunken comportment consists of "time-out" behaviors, subject to the norms of drinking groups, the rewards of such socially licensed behaviors comprising the drinking motive. This perspective was applied to working-class bars in Middletown, Indiana. Although there was a wide range of behavior by bar, drinker category, and the number of drinks consumed, observed patterns suggest the presence of norms. Dramaturgical styles were also noted and typologized; these styles appear to comprise the acting out of sub-culturally desirable roles. The possibility that alcohol facilitates such activities is suggested by earlier laboratory work on drinking fantasies. Drinking rewards may, therefore, stem from altered states of consciousness rather than social license alone.

Requirement for autoinducer in transcriptional negative autoregulation of the Vibrio fischeri luxR gene in Escherichia coli.

The effect of a mutation in luxI (autoinducer synthetase gene) on transcription of luxR in the cloned Vibrio fischeri lux system (luxR, luxICDABE) was examined in Escherichia coli. For the luxI mutant, transcription from the luxR promoter (monitored with beta-galactosidase levels from a luxR::lacZ fusion, with LuxR supplied in trans) decreased fivefold, to levels of the luxI+ strain, only in the presence of added autoinducer. The results demonstrate that, as has been shown at the translational level, autoinducer is required for negative autoregulation of luxR at the transcriptional level.

Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The aroH gene of Escherichia coli, which encodes the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzyme of the common aromatic biosynthetic pathway, was cloned behind the tac promoter in expression plasmid pKK223-3. The enzyme was overexpressed, purified to homogeneity, and characterized. The native enzyme was found to be a dimeric metalloprotein containing 0.3 mol of iron per mol of subunit and variable amounts of zinc. The activity of the native enzyme was stimulated two- to threefold when assayed in the presence of Fe2+ ions. Pretreatment of the enzyme with Fe2+ also resulted in activation, accompanied by an equivalent increase in iron content. Treatment of the enzyme with chelating agents led to inactivation, which was fully reversed by the presence of Fe2+ in the assay mixture. The native enzyme exhibited a unique absorption profile, having a shoulder of absorbance on the aromatic band with a maximum around 350 nm and a broad, weak band with a maximum around 500 nm. Treatment of the enzyme with Fe2+ enhanced the absorbance at 350 nm and eliminated the band at 500 nm. Treatment with reducing agents caused the disappearance of both bands and destabilized the enzyme. Feedback regulation of the activity of the enzyme was specific for tryptophan, with maximum inhibition at about 70%.

The role of matrix metalloproteases and their inhibitors in tumour invasion, metastasis and angiogenesis.

One critical event of tumour invasion that signals the initiation of the metastatic cascade is thought to be interaction of the tumour cell with the basement membrane. Basement membranes may also pose as barriers to tumour cell invasion at multiple points later in the metastatic cascade, including during the processes of vascular infiltration and extravasation. Thus, an important proteolytic event in the metastatic cascade, and also angiogenesis, appears to be degradation of basement membrane components. A specific class of extracellular matrix degrading metalloenzymes, the matrix metalloproteases, and their endogenous inhibitors, the tissue inhibitors of metalloproteases, are thought to have a role in the creation of the proteolytic defect in basement membrane type IV collagen. We will review the evidence which indicates that matrix metalloproteases and tissue inhibitors of metalloproteases are essential for tumour cell invasion and angiogenesis. The regulation of matrix metalloproteases will be discussed, including gene activation and transcription, messenger ribonucleic acid (mRNA) stability, binding of proenzymes to cell membranes and/or matrix components, proenzyme activation, and inactivation by endogenous inhibitors. We will also discuss the mechanism for tissue inhibitor of metalloproteases-mediated inhibition of tumour invasion and angiogenesis. This appears, at least in part, to be through inhibition of protease activity required for cellular invasion, although recent observations suggest that tissue inhibitors of metalloproteases affect other distinct groups of biological activities through mechanisms other than matrix metalloprotease inhibition.