Adrenal chromaffin cells on microcarriers exhibit enhanced long-term functional effects when implanted into the mammalian brain

Rat adrenal chromaffin cells attached to either collagen-coated dextran (Cytodex 3) or glass bead microcarriers, both of 90-200 microns diameter, were used as dopamine-secreting implants in the caudate-putamen of rats with 6-hydroxydopamine-induced unilateral lesions of the substantia nigra. As controls, beads without cells and cells in suspension alone were implanted. Chromaffin cells adhered to microcarriers reduced apomorphine-induced rotation by 75% in lesioned animals. Animals that were lesioned but not receiving cell implants or receiving beads alone showed no reduction. Animals implanted with cells not attached to beads also showed a reduction in rotation but this effect lasted less than three months. Microcarrier-attached cells, however, maintained their effect in reducing rotation for at least eight months (rotations were reduced from a control mean of 10.9 +/- 1.4 to 3.6 +/- 1.1 turns/min) without any "drop-off" of the effect. Histological examination showed that eight months post-implant the cells pre-adhered to beads were still present and could be stained by anti-tyrosine hydroxylase antibody. Sections stained with hematoxylin-eosin showed no signs of an inflammatory response. In contrast to beads implanted into the striatum, Cytodex bead implants injected into the lateral ventricle induced a histopathological response appearing to involve the ependyma and choroid plexus. Results suggest that the striatal parenchyma but not the ventricle is amenable to studies using the microcarrier approach to transplantation.     

Effects of Xenopus laevis mitochondrial single-stranded DNA-binding protein on primer-template binding and 3'-->5' exonuclease activity of DNA polymerase gamma

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.     

Anosognosia in parietal lobe syndrome

Patients with right parietal lesions often deny their paralysis (anosognosia), but do they have "tacit" knowledge of their paralysis? I devised three novel tests to explore this. First, the patients were given a choice between a bimanual task (e.g., tying shoe laces) vs a unimanual one (e.g., threading a bolt). They chose the former on 17 of 18 trials and, surprisingly, showed no frustration or learning despite repeated failed attempts. I conclude that they have no tacit knowledge of paralysis (or, if such knowledge exists, it is not available for this particular task). Second, I used a "virtual reality box" to convey the optical illusion to the patient that she was moving her paralyzed left hand up and down to the rhythm of a metronome, and yet she showed no sign of surprise. Third, I irrigated patient BM's left ear canal with cold water, a procedure that is known to shift that patient's spatial frame of reference by stimulating the vestibular system. Surprisingly, this allowed her "repressed" memory of the paralysis to come to the surface; she said she had been paralyzed continuously for several days. I suggest that the vestibular stimulation produces these remarkable effects by mimicking REM sleep. These patients also employ a whole arsenal of grossly exaggerated Freudian "defense mechanisms" to account for their paralysis. To explain this, I propose that in normal individuals the left hemisphere ordinarily deals with small, local anomalies by trying to impose consistency but, when the anomaly exceeds threshold, an interaction with the right hemisphere forces a "paradigm shift." A failure of this process, in patients with right hemisphere damage, might partially account for anosognosia. Finally, I present a new conceptual framework that may help link several psychological and neurological phenomena such as Freudian defense mechanisms, vestibular stimulation, anosognosia, memory repression, visual illusions, anterograde amnesia, REM sleep, dreaming, and humor.     

Ricin A chain can be chemically cross-linked to the mammalian ribosomal proteins L9 and L10e

Indirect immunofluorescence studies revealed that when fixed, permeabilized cultured human cells were incubated with ricin A chain, the toxin molecule localized in a staining pattern indicative of binding to the endoplasmic reticulum and to nucleoli. Chemical cross-linking experiments were performed to identify the cellular components that mediated the binding of ricin A chain. Conjugates were formed between 125I-labeled ricin A chain and two proteins present in preparations of total cell membranes and in samples of purified mammalian ribosomes. Specificity of the ricin A chain-ribosome interaction was demonstrated by inhibition of formation of the complexes by excess unlabeled ricin A chain, but not by excess unlabeled gelonin, another ribosome-inactivating protein. Complexes of ricin A chain cross-linked to the ribosomal proteins were purified and subjected to proteolytic digestion with trypsin. Amino acid sequencing of internal tryptic peptides enabled identification of the ricin A chain-binding proteins as L9 and L10e of the mammalian large ribosomal subunit.     

Anti-macrophage monoclonal antibody D-11 in the diagnosis of histiocytic tumors

Anti-macrophage monoclonal antibody (Mab) D-11 was tested in surgical material and biopsies of non-epithelial tumors and tumor-like lesions from 181 patients in order to assess possibility of using this Mab for diagnosis of histiocytic tumors, malignant fibrous histiocytoma in particular. The study was performed in parallel on cryostat sections and smears by immuno-peroxidase method. It is established that D-11 reacts positively with both histiocytic tumors and tumors of other genesis this being a limiting factor in differential diagnosis of histiocytic tumors. However, taking into consideration 100% of positive results with histiocytic tumors only, this antibody can be used for exclusion of tumors studied from the group of histiocytomas in cases of negative reaction.     

Analysis of linkage between beta-lactoglobulin and J blood group system loci in cattle

Linkage between loci controlling variants of beta-lactoglobulin and blood groups of the J system in cattle was studied by means of stochastic genetic methods reported earlier. The studies were conducted on a herd of Black Pied cattle improved with Holstein sires; population genetic data were analyzed. A plot for lod score was constructed, and point (r - 0.28) and interval estimations of the coefficient of recombination were obtained. The results are in good agreement with earlier reported data on other subjects.     

Is it possible to perform immediate cardiac assist using untrained latissimus dorsi muscle in a work-rest regimen?

The authors investigated what contractile force (CF) could be obtained from unconditioned latissimus dorsi muscle immediately after mobilization and for the 2 week vascular period of recovery. Latissimus dorsi muscle mobilization was performed on seven adult (4 experimental and 3 control) sheep leaving only the pedicle and the peripheral muscle intact. Telectronics stimulators (Myostim 7220; Teletronics Pacing Systems, Inc, Englewood, CO) were implanted. Immediately after mobilization 11-35% of the initial CF was lost. A 30 min fatigue test was performed 1 hr after mobilization (20 g/kg preload, 10 V, 10 Hz, 15 BPM, 6 impulses per burst) using a 1 min work-1 min rest regimen. Two sheep lost 2-12% of initial CF; two increased CF by 14-24%. At the end of the fatigue test, CF consisted of 74-89% of immobilized CF. Electrical stimulation training of the muscle was then initiated with the following regimen in the experimental animals only: 15 BPM, single impulses, 5 V, 10 Hz. Every day the muscle was exercised using a work-rest regimen to mimic cardiac assist, starting with 20 min on day 2, and increasing by 2 min per day until a total of 50 min was reached on day 16. All animals were retested for CF using a 42 min fatigue test on days 6, 11, and 16. On day 6, there was no fatigue evident in the experimental group during the 42 min test. CF after testing was 59-81% (mean 67%) of initial data. In the control group (animals with no electrical stimulation training protocol), CF decreased by 11% (from 64 to 53%). On day 11, there was no fatigue evident in the experimental group; CF in all animals increased by 2-8%. On day 16, there was also no fatigue evident in the experimental group; CF increased by 0-9%. An additional 20 min of continuous contraction (15 BPM) fatigue testing was performed on the muscle without rest between the tests. No fatigue was evident at the end of testing. Light microscopic analysis of latissimus dorsi muscle biopsy specimens taken on the days of testing showed no evidence of necrotic damage. Our investigations suggest that it may be possible to start muscle transformation immediately after mobilization and use the untrained latissimus dorsi muscle for cardiac assist immediately after surgery for short periods.