Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase A degrades collagen IV but not fibronectin.

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.

Relaxin increases secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 during uterine and cervical growth and remodeling in the pig.

Matrix metalloproteinases are proteolytic enzymes that degrade the extracellular matrix and are essential for tissue remodeling. Uterine and cervical growth require remodeling of structural barriers to cell invasion and matrix metalloproteinase-2 and -9 degrade type IV collagen, the major component of basement membranes. Relaxin stimulates uterine and cervical growth and remodeling, which includes remodeling of support elements such as basement membranes. The objective of this study was to determine whether relaxin alters the production and/or activity of matrix metalloproteinase-2 and -9 in the uterus or cervix of the pig. The growth-promoting effects of relaxin were elicited by administering relaxin to prepubertal gilts every 6 h for 54 h. The expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 was characterized by gel zymography, and proteins were quantified by immunoblotting. Total enzyme activity was measured using matrix metalloproteinase-specific fluorescent substrate assays. In both uterine and cervical tissues, immunoreactive matrix metalloproteinase-2 and matrix metalloproteinase-9 protein expression was similar in relaxin-treated and control animals. However, tissue-associated gelatinase activity was attenuated by relaxin (P < 0.05). In contrast, relaxin significantly increased the secretion of active matrix metalloproteinase-2 and -9 protein into uterine fluid (P < 0.05). Given the importance of matrix metalloproteinases in extracellular matrix degradation, the observation that relaxin promotes uterine secretion of matrix metalloproteinase-2 and -9 supports the concept that relaxin facilitates the growth and remodeling of reproductive tissues by increasing extracellular proteolysis in the pig reproductive tract.

Increased expression of stromelysin 3 mRNA in leiomyomas (uterine fibroids) compared with myometrium.

OBJECTIVE: To determine the level of expression of selected matrix metalloproteinases in uterine leiomyoma compared with unaffected myometrium in an effort to explain the abnormal accumulation of extracellular matrix in the leiomyoma. METHODS: The levels of matrix metalloproteinase (MMP) mRNA in leiomyoma and myometrium were measured in samples from 22 patients during either proliferative (n = 6) or secretory phases (n = 16) of the menstrual cycle. Relative amounts of collagenase (MMP-1) and stromelysin (MMP-3) mRNAs were measured by Northern blot analysis, and amounts of stromelysin 3 (MMP-11) and matrilysin (MMP-7) mRNA from each sample were determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to beta-actin mRNA. RESULTS: The levels of MMP-1 and MMP-3 mRNAs were similar in both leiomyoma and unaffected myometrium. The levels of MMP-11 mRNA were twofold greater in leiomyoma compared with myometrium throughout the menstrual cycle, and the differences in the levels of MMP-11 were significantly different during the secretory phase. The level of MMP-7 mRNA expression was similar in leiomyoma, myometrium, and endometrium. CONCLUSIONS: Among the metalloproteinases examined in this study, only the levels of MMP-11 mRNA were elevated in leiomyoma compared with myometrium. The increased expression of MMP-11 in uterine leiomyoma compared with myometrium is analagous to previously reported elevations of MMP-11 mRNA in dermatofibromas compared with unaffected skin. The increased expression of MMP-11 mRNA in fibroid tumors suggests that this MMP may be involved in the formation of a more fibrous extracellular matrix in leiomyoma relative to unaffected myometrium.

Postural finger tremor exhibited by Parkinson patients and age-matched subjects.

Physiological correlates of postural tremor of the finger seen in Parkinson's disease patients are different from those seen in age-matched control subjects. A significant correlation between the spectral peak of acceleration and the spectral peak of rectified electromyographic activity from the muscle responsible for finger extension was found in Parkinson's disease patients. This correlation was not seen in age-matched control subjects. Any neural drive imposed on the motoneuron pool from supraspinal levels would enhance the electromyographic activity. Likewise, any feedback effects via spinal stretch reflexes or supraspinal stretch responses would be mediated through the motoneuron pool and electromyographic activity. The results of this research support the theory that Parkinson tremor is a centrally driven rhythm that may be influenced by feedback effects, whereas physiological tremor is due to a complex interaction of central, feedback, and mechanical effects.

Seasonal response of the pituitary and testes to gonadotropin-releasing hormone in the black bear (Ursus americanus).

1. The reproductive physiology of the black bear has not been studied extensively. Our objective was to determine if the sensitivity of the pituitary-testes axis to gonadotropin-releasing hormone (GnRH) changes with season. 2. A GnRH dose-response study was conducted using three captive male black bears. Each bear received the same dose of 30, 95 or 300 micrograms GnRH per total body weight in the fall, winter, spring and summer. Blood was sampled at 15-min intervals 1 hr before and 1 hr after GnRH injection and at 30-min intervals during the second hour after injection. Luteinizing hormone (LH) and testosterone were measured in the serum. 3. A heterologous LH radioimmunoassay was established and rigorously validated to measure black bear LH using bovine (b)LH and a monoclonal anti-bLH antibody. 4. Our results suggest: (1) responsiveness of the pituitary to GnRH was highest in the spring and lowest in the winter and (2) pattern of testosterone production is closely correlated with LH released from the pituitary in response to GnRH.

Follicle stimulating hormone increases serum oestradiol-17 beta concentrations, number of growing follicles and yolk deposition in aging hens (Gallus gallus domesticus) with decreased egg production.

1. The aims of this study were to determine if the number of small yellow follicles (SYF) and large white follicles (LWF) in ovaries of young and old hens differed; and if injection of old hens with follicle stimulating hormone (FSH) changed growth of and yolk deposition into follicles of old hens. 2. Ovaries were removed and follicles were divided according to size and condition and counted. The number of normal SYF and LWF was decreased in old hens compared to young hens, whereas the number of atretic follicles was greater in old hens compared to young hens. 3. Old hens were injected subcutaneously with saline containing 0.1% bovine serum albumin (BSA, vehicle) or 12.5, 50, 200, 400 micrograms porcine (p) FSH or 25 or 50 IU pregnant mare's serum gonadotropin (PMSG) for 5 consecutive days. Blood samples were taken on days 1 and 5 before FSH and PMSG injection and 2 h after FSH and PMSG injection on day 5. Sudan black and Sudan red dyes were injected intravenously on alternative days to monitor yolk deposition into follicles of the hierarchy removed after the fifth day of FSH treatment. 4. Treatment with 200 micrograms pFSH or 25 IU PMSG for 5 d increased serum progesterone (P4) concentrations as compared to controls. Injection of hens with FSH caused a linear dose dependent increase in serum oestradiol-17 beta (E2) concentrations, dose dependent increase in SYF and LWF, and dose related decrease in number of atretic SYF and LWF. The hierarchy of the ovary was disrupted with PMSG, but not FSH. Larger doses of FSH increased the number of small follicles (10 mm diameter) and yolk deposition. 5. We conclude that small follicles which have not entered the rapid growth phase are responsive to FSH. The increased yolk deposition following FSH treatment may have been a direct effect of FSH or may have been caused by the elevation of serum E2 concentrations in response to FSH treatment. It is possible that old chickens may produce inadequate amounts of FSH which result in decreased rate of follicular growth and ultimately decreased egg production.

Premovement silence of EMG activity prior to movement in Parkinson patients.

Premovement silence (PMS) of tonic agonist electromyographic activity (EMG) preceding a maximum effort elbow extension was studied in eight bradykinetic Parkinson patients. The occurrence and duration of PMS have been shown to be significantly correlated with peak acceleration of movement in normal subjects. To determine if inability to silence the EMG prior to the initial agonist burst may contribute to bradykinesia of Parkinson's disease, patients maintained elbow extension against a tonic load on triceps, and in response to a tone performed a maximum effort elbow extension. All eight patients showed some trials (mean = 30%) with PMS. However, neither the incidence of PMS nor its duration were significantly correlated with mean peak acceleration. A significant correlation was found between the incidence of abnormal initial agonist bursts, called "segmented bursts," and mean peak acceleration. We conclude that ability to silence the agonist muscle prior to movement is not strongly associated with bradykinesia in Parkinson's disease.

Mitomycin as adjunct chemotherapy with trabeculectomy.

Preliminary experience is reported in using mitomycin to improve the prognosis in trabeculectomy operations considered otherwise likely not to succeed. The medication is applied intraoperatively during an otherwise standard trabeculectomy procedure. Surgery was considered successful if the pressure was lowered to a predetermined target level, the only vision reduction was believed to be on the basis of cataract development, and there was no progression of cupping or visual field loss. With a follow-up of 6 to 42 months, the overall success rate is 84%.

Growth of Dunning transplantable prostate adenocarcinomas in rats fed diets with various fat contents.

The effects of dietary fat concentration (5 vs. 20% corn oil, 0.5 vs. 20% corn oil and fat-free vs. 20% corn oil) on the growth of the Dunning R3327-H (hormone-sensitive, well-differentiated, slow-growing) and R3327-150 (hormone-insensitive, anaplastic, rapidly growing) transplantable prostate tumor sublines were studied in Copenhagen x Fisher F1 male rats. Rats fed 5 vs. 20% corn oil or 0.5 vs. 20% corn oil showed no differences in either R3327-H or R3327-150 tumor growth. Fat-free diets had no effect on the growth of R3327-150 tumors. However, the mean weight of the R3327-H tumor at necropsy, 16 wk after implantation, was reduced by 40% in rats fed a fat-free diet compared with 20% corn oil (P less than 0.05). Energy intake, weight gain and the weight of the pituitary and prostate glands in rats bearing either tumor was lower in rats fed the fat-free diet than in those fed the 20% corn oil diet. There were no differences in serum prolactin, pituitary prolactin or serum testosterone associated with dietary fat concentration in any study. In summary, no differences in transplantable prostate tumor growth were seen over a wide range of lipid intake (0.5-20%), although a fat-free diet reduced the growth of the R3327-H prostate adenocarcinoma.

Annual changes in serum sex steroids in male and female black (Ursus americanus) and polar (Ursus maritimus) bears.

The adaptation of black and polar bears to their environments is proportional to the severity of climate and food restriction. Both black and polar bears mate during the spring, despite differences in their recent metabolic state. Reproductive activity in black bears follows 4 mo of torpor, whereas reproduction in polar bears occurs prior to torpor. The goals of this study were to measure the annual changes in serum sex steroids in male and female black and polar bears, and to determine if changes in serum levels of these steroids were associated with metabolic condition or photoperiod. Serum testosterone (T) concentrations were elevated during spring in black and polar bears. Moreover, this increase in serum T in polar bears during spring was correlated with age and testis size. Serum progesterone (P4) concentrations increased in pregnant polar bears in fall coincident with the time of expected implantation. No increases in serum P4 were observed in nonpregnant black and polar bears. Serum estradiol (E2) was elevated in nonpregnant and pregnant polar bears 2 mo prior to the time of expected implantation. We found that serum sex steroids measured in black and polar bears change independent of torpor. Therefore, our results suggest that photoperiod may be a more important regulator of serum steroid levels and reproduction than metabolic condition.

Premovement silence in agonist muscles preceding maximum efforts.

Electromyographic silent periods (premovement silence) preceding the initial agonist burst were studied in 11 healthy subjects who performed rapid forearm flexions and extensions. Premovement silence occurred with higher frequency during self-paced movements when subjects produced maximum power efforts than during a reaction time paradigm. The occurrence and duration were significantly correlated with the peak acceleration of the movement. Premovement silence occurred specifically in those muscles involved in the intended movement, often in several synergists with different latencies and durations. The shortest latency of premovement silence was considerably briefer than that of the earliest voluntary increase in EMG. The findings suggest that premovement silence may increase peak muscular force by bringing motoneurons into a nonrefractory state prior to their activation. The fact that it occurred on some, but not all, trials within single subjects and had a variable duration from trial to trial suggests that it may be a learned, rather than automatic, motor response.

Intraoperative color transparencies: how to improve your results.

Improvements in film speed and the availability of 55 and 105 mm. macro lenses used with appropriate lens filters have obviated the necessity for flash equipment to obtain high quality intraoperative color transparencies. We have had good results without flash, using only available operating room spotlights with a lens/film/filter combination readily available to the surgeon. We present this system and the rationale for its effectiveness.

Exercise therapy for Parkinson's disease.

The outcomes of two different 12-week exercise programs were assessed by machine measurements of motor signs, tests of grip strength, motor coordination and speed, and neurophysiologic determinations of long-latency stretch responses in two groups of Parkinson patients matched for age, sex and stage of disease. The programs tested included an exercise program developed by the United Parkinson Foundation and a program of upper body karate training. Outcomes of these programs were similar. The majority of patients in both groups showed improvements in gait, tremor, grip strength and motor coordination on tasks requiring fine control. In one task involving whole body coordination there was a decline in function, while muscle rigidity was unchanged. The findings suggest that exercise is a useful adjunct to pharmacologic therapy.

Effects of single intracortical microstimuli in motor cortex on activity of identified forearm motor units in behaving monkeys.

We examined the magnitude and extent of output effects elicited from focal cortical sites on the activity of individual motor units (MUs) by delivering single-pulse intracortical microstimuli (S-ICMS) (5-15 microA) during isometric wrist activity. Stimulation sites in the precentral gyrus (area 4) were chosen for study if stimulus-triggered averages (stimulus-TAs) of multiunit electromyograms (EMGs) revealed poststimulus facilitation (PStimF) of EMG activity in any of the coactivated wrist muscles. Single MUs were then isolated in the facilitated muscles with a remotely controlled tripolar microelectrode. MUs were identified by their signatures in their parent muscles (from MU-triggered averages of EMGs) and by their firing pattern during ramp-and-hold wrist responses. One objective was to quantify the magnitude and time course of the effects on single MUs by compiling peristimulus histograms of MU firing. The cross-correlation histograms between S-ICMS and MU action potentials showed peaks with onset latencies of 8.8 +/- 1.7 ms (mean +/- SD, n = 64) and durations of 1.8 +/- 1.2 ms (n = 104). The cumulative sums of the correlogram peaks resembled the rising phase of corticomotoneuronal excitatory postsynaptic potentials previously recorded in forelimb motoneurons. Comparison of correlogram peaks with stimulus-TAs of MU potentials suggests that the duration of PStimF of multiunit EMG can be accounted for, in approximately equal proportions, by l) the variation in firing time of single MUs (i.e., the width of the MU correlogram peaks), 2) the width of single MU potentials, and 3) the contribution of different MUs at different latencies. The sizes of the correlogram peaks relative to base line were larger than the PStimF of multiunit EMGs, and increased more rapidly with stimulus intensity, indicating appreciable cancellation in the multiunit records. A second objective was to determine whether S-ICMS affected all the MUs of a facilitated muscle, or only a particular subset. Of 104 MUs sampled in facilitated muscles, 99 (95%) were found to be individually facilitated (P less than 0.05). MU firing patterns during isometric ramp-and-hold torque responses were characterized as phasic, phasic-tonic, tonic, or decrementing; stimulation at a given cortical site was found to facilitate all four types of MUs. When more than one muscle showed PStimF from a site, MUs belonging to each of the facilitated muscles were facilitated individually by S-ICMS at that site.(ABSTRACT TRUNCATED AT 400 WORDS)

Discharge properties of primate forearm motor units during isometric muscle activity.

Activity of single motor units (MUs) was recorded in forelimb muscles of rhesus macaques while they generated isometric ramp-and-hold torques about the wrist. Multiunit electromyographic (EMG) activity was recorded from 10-12 identified flexor and extensor muscles of the wrist and digits with implanted EMG wire electrodes. Single MUs from these muscles were recorded with a remotely controlled tripolar microelectrode array. The parent muscle of each MU was determined by compiling MU-triggered averages of multiunit EMGs. The MU firing patterns during the isometric task were determined from response histograms aligned with change in torque. At moderate torque levels, MUs (n = 86) exhibited four types of discharge patterns during the ramp-and-hold trajectory: phasic-tonic (23%), tonic (33%), decrementing (39%), and phasic (5%). Phasic-tonic MUs exhibited a phasic burst of activity during the torque ramp which exceeded the firing rate during the static hold period. Both phasic-tonic and tonic MUs exhibited a constant mean firing rate during the hold period; the discharge of decrementing MUs gradually decreased during the static hold. Phasic MUs fired only during the change in force. The relation between MU firing rate and torque was investigated as the monkeys generated responses of different levels of static torque during the hold period. Mean firing rate during the hold was found to be proportional to static torque up to moderate torque levels, where it reached a maximum. In the linear range, the mean rate-torque slope was 3.4 +/- 1.9 imp/s per 10(5) dyn . cm (n = 9).

Hormone-stimulable adenylyl cyclase and steroid concentration of follicles of the pregnant mare's serum gonadotropin-treated hen.

Pregnant mare's serum gonadotropin (PMSG) treatment of the hen disrupts the follicular hierarchy and causes cessation of ovulation. We measured serum progesterone (P4) and estradiol (E2) concentrations and follicular steroid levels and adenylyl cyclase (AC) activity of PMSG-treated hens. Serum P4 and E2 levels were elevated (P less than 0.01 and P less than 0.05, respectively) in PMSG-treated hens compared to controls. There was no significant difference in P4 and E2 concentrations in granulosa and theca layers, respectively, between follicles from PMSG-treated hens and the largest (F1) follicles from control hens. Basal, luteinizing hormone (LH)-, and follicle-stimulating hormone (FSH)-stimulable AC activity was measured in granulosa layers of the largest follicles from PMSG-treated hens and the F1 and second largest (F2) follicles from control hens. Basal AC activity was increased in follicles from PMSG-treated hens (P less than 0.05) compared to F1 control follicles. There was no difference in LH- and FSH-stimulable AC of PMSG-treated hens compared to F1 controls. Control F2 follicles had lower LH- (P less than 0.001) and FSH-stimulable (P less than 0.005) AC activity than follicles from control F1 or PMSG-treated hens. Relative LH- and FSH-stimulable AC (hormone stimulable vs. basal) for follicles from PMSG-treated hens did not differ statistically from the relative AC activity of vehicle-injected F1 or F2 follicles. Therefore, in spite of the high serum P4 and E2 levels in the PMSG-treated hens, there was no change in the hormone-stimulable AC system.(ABSTRACT TRUNCATED AT 250 WORDS)