Use of 125I-secretin to identify and characterize high-affinity secretin receptors on pancreatic acini

We prepared 125I-secretin and studied the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini prepared from guinea pig pancreas. Iodinated secretin retained intrinsic biological activity in that it was as effective but 2.5-times less potent than native secretin in its ability to bind to pancreatic acini and to increase cellular cAMP. Scatchard analysis of binding of 125I-secretin indicated that each pancreatic acinar cell has approximately 93,000 binding sites, half of which are occupied by 11 nM iodinated secretin. Binding of 125I-secretin was rapid, reversible, saturable, specific, and temperature dependent. Binding of 125I-secretin was inhibited by secretin, vasoactive intestinal peptide, PHI, and Gila monster venom but not by glucagon, gastric inhibitory polypeptide, cholecystokinin, caerulein, gastrin, bovine pancreatic polypeptide, somatostatin, neurotensin, leucine-enkephalin, methionine-enkephalin, carbachol, bombesin, litorin, eledoisin, physalaemin, or substance P. With agonists (secretin, vasoactive intestinal peptide, PHI, or Gila monster venom), as well as antagonists (C-terminal fragments of secretin), there was a close correlation between their relative potencies for inhibiting binding of 125I-secretin and their relative potencies for increasing cAMP (agonists) or inhibiting the secretin-induced increase in cAMP (antagonists). For a given agonist, however, a 40-fold higher concentration was required for half-maximal inhibition of binding of 125I-secretin than was required to produce a half-maximal increase in cellular cAMP. Thus, maximal stimulation of cellular cAMP occurs when approximately one-third of the secretin receptors are occupied by an agonist.     

Transient and sustained types of long-term potentiation in the CA1 area of the rat hippocampus

Synaptic potentiation induced by high frequency stimulation was investigated by recording field excitatory postsynaptic potentials (f-EPSPs) in rat hippocampal slices. Potentiation consisted of a transient period of decaying f-EPSPs (short-term potentiation, STP) that led to a plateau of continuously potentiated f-EPSPs (long-term potentiation, LTP). Here we show that a previously unknown type of transient, use-dependent, long-lasting potentiation (t-LTP) can account for STP. t-LTP could be stored for more than 6 h and its decay was caused by synaptic activation. Both the expression and the decay of t-LTP were input specific. t-LTP was induced differently from conventional LTP in that the amplitude of t-LTP was dependent upon the stimulation frequency, whereas the magnitude of LTP was dependent on the number of stimuli in the induction train. The decay of t-LTP could not be prevented by the blockage of glutamate receptors, but was prevented by the blockage of stimulus-evoked neurotransmitter release, suggesting that t-LTP is expressed presynaptically. Paired-pulse stimulation experiments showed that the decay of t-LTP was mediated by a decrease in the probability of neurotransmitter release. The decline of t-LTP could be prolonged by the activation of NMDA receptors. Hence, both single and paired-pulse stimuli prolonged the decline of the t-LTP. This decline could be prevented by high frequency burst stimulation (200 Hz). We conclude that t-LTP allows dynamic modulation of synaptic transmission by providing not only spatial association but also temporal convergence between synaptic inputs. Therefore, t-LTP might be a substrate for the encoding of synaptic memory.     

Histopathological grading of soft tissue tumours. Prognostic significance in a prospective study of 278 consecutive cases

A consecutive 10-year series of 278 soft tissue sarcomas was prospectively graded, using a system based on the number of mitoses and taking into account parameters such as cellularity, anaplasia, necrosis, and histogenetic type and subtype of tumour. Prognostic factors in relation to metastasis-free survival were studied by uni- and multivariate analysis. Fifty-seven (20.5 per cent) were low-grade tumours, 43 (15.5 per cent) were intermediate, and 178 (64 per cent) were high grade. High-grade tumours were divided into two groups; 80 (29 per cent) grade 3A (= 5-20 mitoses per 10 high power fields (HPF)) and 78 grade 3B (28 per cent) (= more than 20 mitoses/10 HPF); 10 HPF corresponds to 2.5 mm2. Twenty (7.2 per cent) high-grade tumours could not be further subdivided. Grading was found to be the prognostic factor associated with the strongest predictive value. Five-year survival in low-grade and intermediate tumours (95 and 86 percent, respectively) differed significantly (P less than 0.0001) from high grade (50 per cent) and (p = 0.0018) between grade 3A (64 per cent) and grade 3B (41 per cent). Other prognostic indicators of importance in high-grade tumours were age, local recurrence at presentation (primary operation outside the Centre), and localization (superficial vs. deep).     

The vascularization of the hypothalamic-hypophyseal region of the eastern brook trout,Salvelinus fontinalis

The hypophysis of the brook trout is irrigated by blood vessels which originate from the internal carotid arteries. These are: (1) branches of the ventral hypothalamic arteries that give rise to an extensive capillary plexus in the neurohypophysis from which vessels extend into all regions of the adenohypophysis; (2) branches of the caudal hypothalamic artery that irrigate the saccus vasculosus and continue anteriorly to supply the ventral areas of the meta-adenohypophysis; (3) a caudal hypophyseal artery which vascularizes a portion of the meta-adenohypophysis however, this vessel is not always present; (4) a pair of small arteries which supply the peripheral regions of the gland directly from the carotids. Most of the neurosecretory fibers of the preoptic-hypophyseal tract terminate close to capillaries in the neurohypophysis. A few axons extend into meso-adenohypophyseal tissue. It is suggested that the secretory activities of the pro-, meso- and metaadenohypophyses are governed by hypothalamic factors that are chiefly transmitted to the gland cells via the vascular system (indirect control). However, the activity of the meso-adenohypophysis may also be regulated by factors which are transmitted directly to the cells from the endings of neurosecretory fibers which have traversed the neurohypophysis (direct control). The distribution and abundance of neurosecretion in the ventral hypophysis suggest the possibility of storage of hypothalamic products within this region.     

CXCR4 and CCR5 expression on CD4+ T cells in vivo and HIV-1 antigen beta-chemokine production in vitro after treatment with HIV-1 immunogen (REMUNE)

BACKGROUND: The chemokine receptors CXCR4 and CCR5 have been identified as the major coreceptors for HIV-1 on CD4+ cells and macrophages. The natural ligands for these receptors are SDF-1 and the beta-chemokines (MIP-1alpha, MIP-1beta, RANTES), respectively, and are the products of a variety of immune cells, including CD8+ T lymphocytes. STUDY DESIGN/METHODS: We hypothesized that the ability to stimulate the natural ligands for these receptors using an immune based therapy might influence in vivo chemokine receptor expression. RESULTS: In vivo CXCR4 expression remained stable after treatment with an HIV-1 Immunogen (REMUNE), whereas CCR5 expression on CD4+ T cells decreased (p < .05). Furthermore, HIV-1 antigen-specific production of beta-chemokines in vitro was also augmented (P < .05). CONCLUSIONS: These preliminary results suggest that this HIV-1-specific immune-based therapy can stimulate antigen-specific beta-chemokine production in vitro and downregulate CCR5 receptor expression on CD4 cells in vivo.     

Expansion of CD5 - B cells in multiple sclerosis correlates with CD80 (B7-1) expression

The pathogenetic role of autoantibodies in multiple sclerosis (MS) is uncertain. CD5+ B cells commonly produce autoantibodies, but CD5 expression has also been implicated in B-cell tolerance. We studied B-cell subsets, anti-myelin protein antibody-secreting cells in cerebrospinal fluid (CSF) and a panel of serum autoantibodies in patients with clinically isolated syndromes (CIS), suggestive of MS and patients with clinically definite MS (CDMS). Patients with CDMS had a higher percentage of CD5- B cells in CSF than did control subjects (P = 0.02). CIS patients with immunoglobulin G (IgG) oligoclonal bands in CSF or multiple lesions on magnetic resonance imaging (MRI) had a higher percentage of CD5- B cells in CSF than did the remaining CIS patients (P = 0.03). The percentage of CD5- and CD80+ B cells correlated positively and the percentage of CD5+ B cells correlated negatively with the number of CSF cells secreting anti-myelin basic protein (anti-MBP) antibodies. The prevalence of serum autoantibodies was comparable in the three patient groups. We conclude that intrathecal expansion of CD5- B cells appears to be more characteristic in MS patients, and CD5+ B cells may be associated with a lower prevalence of anti-myelin antibody production.     

Gentamicin susceptibility in Escherichia coli related to the genetic background: problems with breakpoints

In total, 120 Escherichia coli isolates positive for one of the gentamicin resistance (GEN(R)) genes aac(3)-II, aac(3)-IV or ant(2')-I were tested for gentamicin susceptibility by the agar dilution method. Isolates positive for aac(3)-IV or ant(2')-I had an MIC distribution of 8-64 mg/L, whereas isolates positive for aac(3)-II had MICs of 32 to >512 mg/L, suggesting a relationship between the distribution of MICs and the specific GEN(R) mechanism. The MIC distribution, regardless of the GEN(R) mechanism, was 8 - >512 mg/L, which supports the clinical breakpoint of MIC >4 mg/L suggested by EUCAST and questions the breakpoint recommended by the CLSI (> or =16 mg/L).     

Motor unit recruitment and rate coding in response to fatiguing shoulder abductions and subsequent recovery

The purpose of the present study was to investigate motor unit (MU) recruitment and firing rate, and the MU action potential (MUAP) characteristics of the human supraspinatus muscle during prolonged static contraction and subsequent recovery. Eight female subjects sustained a 30° shoulder abduction, requiring 11–12% of maximal voluntary contraction (MVC), for 30 min. At 10 and 30 min into the recovery period, the shoulder abduction was repeated for 1 min. The rating of perceived exertion for the shoulder region increased to “close to exhaustion” during the prolonged contraction, and the surface electromyography (EMG) recorded from the deltoid and trapezius muscles showed signs of local muscle fatigue. From the supraspinatus muscle, a total of 23,830 MU firings from 265 MUs were identified using needle electrodes. Of the identified MUs, 95% were continuously active during the 8-s recordings, indicating a low degree of MU rotation. The mean (range) MU firing rate was 11.2 (5.7–14.5) Hz, indicating the relative force contribution of individual MUs to be larger than the overall mean shoulder muscle load. The average MU firing rate remained stable throughout the prolonged abduction, although firing rate variability increased in response to fatigue. The average concentric MUAP amplitude increased by 38% from the beginning (0–6 min) to the end (24–29 min) of the contraction period, indicating recruitment of larger MUs in response to fatigue. In contrast, after 10 min of recovery the average MU amplitude was smaller than seen initially in the prolonged contraction, but not different after 30 min, while the MU firing rate was higher during both tests. In conclusion, MU recruitment plays a significant role during fatigue, whereas rate coding has a major priority during recovery. Furthermore, a low degree of MU rotation in combination with a high relative load at the MU level may imply a risk of overloading certain MUs during prolonged contractions. Accepted: 6 June 2000     

Identification of recurrent and novel mutations in the LDL receptor gene in Spanish patients with familial hypercholesterolemia. Mutations in brief no. 135. Online

We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(-18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706-10G->A and 2390-1G->A, and the other one has been reported recently, 313+1G->C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G->C; C95R and D679E; W(-18)X and E256K, and C281Y and 1706-10G->A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population.