Cytomechanics of axonal development

Mechanical tension is a robust regulator of axonal development of cultured neurons. We review work from our laboratory, using calibrated glass needles to measure or apply tension to chick sensory neurons, chick forebrain neurons, and rat PC12 cells. We survey direct evidence for two different regimes of tension effects on neurons, a fluid-like growth regime, and a nongrowth, elastic regime. Above a minimum tension threshold, we observe growth effects of tension regulating four phases of axonal development: 1. Initiation of process outgrowth from the cell body; 2. Growth cone-mediated elongation of the axon; 3. Elongation of the axon after synaptogenesis, which normally accommodates the skeletal growth of vertebrates; and 4. Axonal elimination by retraction. Significantly, the quantitative relationship between the force and the growth response is surprisingly similar to the simple relationship characteristic of Newtonian fluid mechanical elements: elongation rate is directly proportional to tension (above the threshold), and this robust linear relationship extends from physiological growth rates to far-above-physiological rates. Thus, tension apparently integrates the complex biochemistry of axonal elongation, including cytoskeletal and membrane dynamics, to produce a simple "force input/growth output" relationship. In addition to this fluid-like growth response, peripheral neurons show elastic behaviors at low tensions (below the threshold tension for growth), as do most cell types. Thus, neurites could exert small static forces without diminution for long periods. In addition, axons of peripheral neurons can actively generate modest tensions, presumably similar to muscle contraction, at tensions near zero. The elastic and force-generating capability of neural axons has recently been proposed to play a major role in the morphogenesis of the brain.     

Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors

Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. We purified the enzyme from the murine colon 26 tumor using a two-step procedure through 5-amino-benzylacyclouridine affinity chromatography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. The polyclonal antibody used to screen a human liver expression library allowed the isolation of a 1.2-kb clone that contained the entire open reading frame of the human UPase. The UPase cDNA has been expressed as a fusion protein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demonstrated that the recombinant UPase preferentially uses uridine, 5-fluorouracil, and uracil as substrates, although lower levels of activity were observed with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues were assayed for phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Activity in normal tissues appeared to be low but very sensitive to BAU (approximately 90% inhibition at 10 microM). Tumors had generally 2-3-fold greater activity compared with adjacent normal tissues. In breast cancer specimens and head-neck squamous carcinomas, however, uridine cleavage was only partially inhibited (40-60%) by 10 or 100 microM BAU. The BAU-insensitive activity requires phosphate and pH conditions similar to the normal enzyme, and the new phosphorolytic activity was independent from thymidine phosphorylase. The BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosphorylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors.     

Clinicopathologic analysis of microscopically invasive breast carcinoma

Breast biopsy or mastectomy cases having diagnoses of carcinoma in situ with "microinvasion," "minimal invasion," "focal invasion," or "suggestive of invasion" were reviewed and all histologically identified foci of invasive disease from each case were measured using an ocular micrometer. Cases in which any single focus of invasion was greater than 5 mm or the added size of separate invasive foci exceeded 10 mm were excluded, resulting in a study group of 75 patients. Invasive neoplasm was present in the initial biopsy in 69 of 75 cases (92%); however, residual invasive neoplasm was found in the subsequent lumpectomy/mastectomy from 14 of these (20%). In 59% of cases, two or more histologically separate foci of invasion were identified. Invasive foci consisted of isolated cells or cell clusters, each less than 1 mm (microfocal invasion), in 33% of cases. In 12 cases, the sum of individual invasive foci was 5 to 10 mm. Axillary lymph nodes (LN) from 5 of 69 patients (7%) contained metastatic carcinoma (four cases, one LN positive; one case, two LN positive). The cumulative sizes of all invasive foci in the LN-positive group were microfocal invasion (one case), 0.6 mm (one case), 1.1 mm, 2.5 mm, and 5.8 mm. The difference in frequency of axillary node metastasis between tumors with microfocal and measurable invasion (4.3% v 8.6%) was not statistically significant. Follow-up data were available on 55 cases (mean interval, 66.1 months). One (node-negative) patient had duct carcinoma in situ recurrence in the same breast 4 years after initial treatment. Another (with unknown node status) developed an axillary lymph node metastasis 13 months after initial treatment (96% disease-free survival). We conclude that microscopic stromal invasion in breast carcinoma, at least in the setting of significant in situ component, is often initiated from multiple foci. Patients with microscopically invasive breast carcinoma have a small but significant risk of axillary metastases, although a highly favorable survival.     

Proline isomerization-independent accumulation of an early intermediate and heterogeneity of the folding pathways of a mixed alpha/beta protein, Escherichia coli thioredoxin

Oxidized Escherichia coli thioredoxin (Trx) is a small protein of 108 residues with one disulfide bond (C32-C35 essentially involved in the activity) and no prosthetic moieties, which folds into a structural motif containing a central twisted beta-sheet flanked by helices that is found in many larger proteins. The kinetics of refolding of Trx in vitro have been investigated using a newly developed active site titration assay and continuous or stopped-flow (SF) methods in conjunction with circular dichroism (CD) and fluorescence (Fl) spectroscopy. These studies revealed the presence of early folding intermediates with "molten globule or pre-molten globule" characteristics. Measurements of the ellipticity at 222 nm indicated that about 68% of the total change associated with refolding occurred during the dead time (4 ms) of the stopped-flow instrument, suggesting the formation of substantial secondary structure. The reconstruction of the far-UV CD spectrum of the burst intermediate using combined continuous and stopped-flow methods showed the formation of a defined secondary structure that contains more beta-structure than the native state. Kinetic measurements using SF far-UV CD and Fl over a wide range (0.087-6 M) of GuHCl concentrations at two temperatures (6 and 20 degreesC) demonstrated that the population formed during the 4 ms dead time contained multiple species that are stabilized mainly by hydrophobic interactions and undergo further folding along alternative pathways. One of these species leads directly and rapidly to the native state as demonstrated by active site titration, while the two others fold into a fourth intermediate that is slowly converted to the native protein. Double-jump experiments suggest that the heterogeneity in folding behavior results from proline isomerizations occurring in the unfolded state. Conversely, the accumulation of the burst intermediate does not depend on proline isomerizations.     

Developments in the treatment of alcoholism

The treatment of alcoholism has changed during the past 2 decades. Notable developments have occurred in pharmacotherapy, psychotherapy, and health-care delivery. A better understanding of the biologic basis for addiction has led to clinical trials of medications that target neuroreceptors. One such medication is the opiate antagonist naltrexone, which decreases the craving for alcohol. Psychosocial interventions continue to be the mainstay of alcohol treatment programs. The efficacy of three different therapies was demonstrated in a study called Project MATCH (Matching Alcoholism Treatments to Client Heterogeneity). This study, however, did not prove the patient-treatment "matching" hypothesis. In addition to therapies provided by addiction specialists, interest is growing in the use of brief motivational techniques in primary-care settings. As the field of addiction responds to an unfolding health-care delivery system, a broader range of treatment options in conjunction with a greater opportunity to individualize patient care is evolving.     

Flap prefabrication: effectiveness of different vascular carriers

A new experimental model of a vascular carrier to prefabricate a "secondary" island flap, the popliteal musculovascular pedicle, was developed in the rat. Using quantitative skin-surface fluorometry 30 minutes after sodium fluorescein injection and a flap survival area in the prefabricated 8 x 2.5-cm abdominal composite island flap, we compared the revascularization ability of our muscular carrier to nonrevascularized controls: the skeletonized arteriovenous pedicle and the fasciovascular pedicle. The free composite graft with no vascular carrier exhibited near-total necrosis. The skeletonized vascular pedicle demonstrated 15.2% +/- 7.8% perfusion of normal skin on dye fluorescence index measurements and 50% flap survival. The fasciovascular pedicle exhibited better revascularization, with a dye fluorescence index of 36.2 +/- 15.5 (p < 0.01) and 90% +/- 10% flap survival (p < 0.001). India ink injection study and histological examination of our model provided visual evidence of revascularization from the musculovascular pedicle, along with preservation of the carrier's muscular architecture. The musculovascular pedicle is a reliable carrier for making new, vascularized composite flaps.     

The gradual threshold model of ambivalence: relating the positive and negative bases of attitudes to subjective ambivalence

Eighty subjects viewed and visually imagined upright or rotated alphanumeric characters and later judged whether test characters were previously seen or imagined (reality monitoring). Identification and test characters were presented verbally or visually. When characters were identified and tested verbally, source confusions (misjudging a seen character as "imagined" and vice-versa) were infrequent and were comparable for rotated and upright characters. When characters were identified and tested visually, source confusions were more frequent and were influenced by character rotation. Memories for imagined characters were especially susceptible to source confusion. Also source confusions for seen characters increased when characters were rotated. These results are consistent with the proposal that increasing sensory similarity between perceived and imagined items increases source confusion and that perceived rotation generates cognitive operations similar to those generated when the subject imagines a character rotated.     

Fast transmission CT for determining attenuation maps using a collimated line source, rotatable air-copper-lead attenuators and fan-beam collimation

We describe a technique using a line source and a rotatable air-copper-lead assembly to acquire gamma transmission computed tomographic (TCT) data for determining attenuation maps to compensate SPECT emission scans. The technique minimizes problems associated with discriminating 99mTc transmission and 201Tl emission photons and requires only a modest increase in total study time. A 99mTc line source and a stacked foil ("multislat") collimator are placed near the focal line of a fan-beam collimator (114 cm focal length) mounted on one detector of a triple-camera SPECT system. We acquired TCT data of plastic rod and anthropomorphic thorax phantoms to investigate the capability of the line source and rotatable air-copper-lead attenuators to determine attenuation maps. The data were acquired with and without 5.4 MBq (145 microCi) of 201Tl placed in the myocardial chamber of the thorax phantom. Phantoms also were scanned using a curved transmission slab source mounted to a parallel-hole collimator. Fan-beam TCT images have improved resolution compared with parallel-beam TCT images. Two patient scans also were performed to evaluate the clinical usefulness of fan-beam TCT. The rotatable air-copper-lead attenuator method eliminates contamination of emission data by transmission photons and reduces spill-over of emission data into the transmission energy window for some cases. Results show the feasibility of using fast, sequential or interlaced transmission scans of a line source within a rotatable air-copper-lead attenuator assembly to obtain accurate attenuation maps for SPECT attenuation compensation.     

Laparoscopic vs. open wedge biopsy of the liver

The number of people smoking free-base cocaine, or "crack," has increased dramatically in recent years. Concomitantly, the literature describing complications of such use has grown as well. Adverse pulmonary effects are being increasingly noted, such as respiratory symptoms, pulmonary hemorrhage, pulmonary edema, asthma, and pulmonary barotrauma. These and other pulmonary effects are reviewed.     

Adaptation of thermafil components to canal walls

In summer 1991 an outbreak of a Salmonella enteritidis epidemic involving about 600 cases of gastroenteritis occurred at one of the leading pharmaceutical companies in southwestern Germany. The main source was a cold fruit soup, in addition Salmonella were isolated from meat strips and a curd cheese which were used for a salad dressing. A total of 2300 contaminated food portions were served resulting in an attack rate of about 25%. The possible origin could have been an asymptomatic Salmonella-positive member of the kitchen personnel who was the only one who was involved with the preparation of all the incriminated foods. A further spread of the epidemic and especially the possible contamination of pharmaceuticals was avoided by the timely and adequate reaction of the company's occupational medical service. This case exemplifies how classical crisis management, "increased initiative on one's own for prevention of infections in all areas of food processing" (Steuer) and finally the cooperation of the company with different institutions of the public health authorities contribute to the control of such a catastrophic scenario.