Cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived lambda gt11 expression library

We report here the cloning of the cDNA coding for platelet connective tissue-activating peptide-III (CTAP-III) from a lambda gt11 expression library prepared using messenger RNA (mRNA) isolated from human platelets. The open reading frame of the clone coded for a protein with 128 amino acid residues. Since the precursor of CTAP-III, platelet basic protein (PBP is 94 amino acids long, the 5'-translated region of the cDNA codes for a leader sequence 34 amino acids long. This leader sequence, like the sequence of mature CTAP-III, shows significant homology to the sequence of platelet factor 4 (PF4), the only other platelet specific alpha-granule protein cloned until now, from a human erythroleukemic (HEL) cell line-derived cDNA library. These leader sequences are probably critical for targeting such proteins to the alpha-granule. Northern blot hybridization with platelet and megakaryocyte mRNA shows a single species mRNA of approximately 0.8 kb, suggesting that the corresponding cDNA is full length. The cloning of platelet specific CTAP-III provides additional evidence for the platelet specificity of the cDNA library used.     

Refinement of a rodent model of peripheral lymphedema.

A reliable, inexpensive experimental counterpart of peripheral lymphedema has been notoriously difficult to reproduce thereby stifling basic and clinical research into this frustrating clinical condition. Accordingly, in 45 adult Wistar-Fuzzy rats, we attempted to produce sustained hindlimb lymphedema by either groin nodal/lymphatic microsurgical ablation (S) (guided by visual blue dye lymphography) or limited field-groin irradiation (R) alone (4500 rads) or combined S followed by R or R followed by S with an additional non-manipulated group serving as controls. Observations were made for 30-100 days thereafter. Hindlimb volumes were determined serially using the truncated cone formula based on multiple circumferential measurements at standardized intervals along the affected hindlimb and the findings compared with similar measurements in the contralateral non-manipulated hindlimb. In randomly selected rats from each group, lymphatic drainage was assessed by lymphangioscintigraphy (LAS), soft tissue swelling by magnetic resonance imaging (MRI), and edema fluid total protein content by refractometry. Whereas S or R alone produced only transient or mild hindlimb edema without associated morbidity or mortality, S-R or R-S induced moderate to severe sustained protein-rich hindlimb lymphedema associated with 9-13% early mortality and notable late local limb morbidity. Lymphatic obstruction was documented by sustained maintenance of increased hindlimb volume, subcutaneous fluid accumulation (MRI), and impaired lymphatic drainage (LAS). This reproducible rodent model of secondary lymphedema reliably simulates a stable clinical condition for a window of up to 100 days and should thereby facilitate standardized testing of therapeutic/preventive protocols and basic research into lymphatic dynamics in secondary lymphedema.     

Neuromedin B and gastrin-releasing peptide mRNAs are differentially distributed in the rat nervous system

The bombesin-like peptides are a family of structurally related amidated peptide ligands that are known to have a variety of potent pharmacological actions on various cells, including neurons in the rat brain. Two mammalian representatives of the bombesin family of peptides have been identified, gastrin-releasing peptide (GRP) and neuromedin B (NMB). Previously, we cloned the rat preproGRP gene and determined the locations of neurons expressing this gene using in situ hybridization. In this study, we describe the structure and sequence of the rat preproNMB gene, and the first detailed cellular localization of preproNMB mRNA in rat brain using in situ hybridization. Nucleotide sequence analysis of cDNA and genomic clones reveals a 117 amino acid precursor whose overall structure is similar to that described for human preproNMB. Sequence similarity between the rat NMB and GRP genes is observed only over a limited 10 amino acid sequence encoding the carboxy termini of the GRP and NMB peptides, the region shown to be necessary and sufficient for high-affinity receptor binding. In situ hybridization studies performed with cRNA probes specific for NMB or GRP mRNA show that the distribution of cells expressing either mRNA in brain is very distinct. NMB mRNA is found most prominently in the olfactory bulb, dentate gyrus, and dorsal root ganglion. In contrast, the highest levels of GRP mRNA are observed in the forebrain (isocortex and hippocampal formation). This heterogeneity of mRNA distribution for these peptides suggests that these 2 structurally related peptides may have very distinct functions as neuropeptides in the rat nervous system.     

Cardiovascular effects of volatile anesthesia in rabbits: influence of chronic heart failure and enalaprilat treatment.

Circulatory responses to isoflurane and halothane anesthesia were studied in eight rabbits with biventricular cardiomyopathy induced by doxorubicin (Adriamycin, 14 mg/kg IV over 7 wk) and in eight controls (saline injections). In preliminary operations pulsed-Doppler flow probes were placed on the ascending aorta, left renal artery, and lower abdominal aorta. Each group was studied after 4, 6, and 7 wk of treatment. The development of congestive heart failure (CHF) was associated with decreases in mean arterial pressure and cardiac output (CO) of 14% and 16%, respectively, (P less than 0.05) and an increase in heart rate. In controls, each anesthetic agent produced dose-related decreases in mean arterial pressure and increases in heart rate, but not significant changes in CO. Renal blood flow was reduced to a similar degree by 1.3 MAC halothane (24% decrease) and 1.3 MAC isoflurane (21% decrease); hindlimb blood flow was reduced only by halothane. As CHF developed there was an attenuation of the heart rate response to anesthesia. Halothane, but not isoflurane, significantly reduced CO in more advanced stages of CHF. The changes in renal blood flow and hindlimb blood flow with each anesthetic in the CHF group were similar to those observed in controls and did not vary with week of treatment. Administration of the angiotensin-converting enzyme inhibitor enalaprilat (0.2 mg/kg IV) reversed the CO and renal blood flow effects of halothane except after 7 wk of treatment in the CHF group, when the combination of halothane and enalaprilat resulted in severe circulatory depression.     

Use of seclusion and restraint in public psychiatric hospitals: patient characteristics and facility effects

Use of seclusion and restraint in 23 adult public psychiatric hospitals in New York State was examined by comparing demographic and diagnostic characteristics of 657 patients who were secluded or restrained during a four-week period with characteristics of 22,939 patients who were not. Logit analysis was used to calculate the probability of seclusion and restraint of individual patients with various combinations of characteristics. Characteristics associated with high probabilities included age less than 26 years, length of stay from 30 to 365 days, involuntary legal status, female gender, a diagnosis of mental retardation, and residence in a hospital with a high rate of seclusion and restraint. The rate of seclusion and restrain in the 23 hospitals ranged from .4 to 9.4 percent of patients. Both patient characteristics and the hospital of residence were needed to explain the case-by-case probability of a patient's being secluded or restrained.     

Postoperative hypoxemia after nonabdominal surgery: a frequent event not caused by nitrous oxide

We tested whether anesthesia that includes nitrous oxide (N2O) results in the development of intraoperative and postoperative pulmonary complications, including hypoxemia. We also tested whether aging contributes to the development of such complications, particularly when anesthesia includes N2O. We randomly allocated patients having total hip replacements, carotid endarterectomies, or transsphenoidal hypophysectomies (total n = 270) to an anesthetic regimen with and without N2O (stratified within surgical group). A heat-and-moisture exchanger was included in the anesthetic circuit of all patients. Patients were monitored perioperatively and for 1 wk after surgery using intermittent and continuous pulse oximetry to determine oxyhemoglobin saturation. Intraoperatively, mean oxygen (O2) saturations were lower in patients given N2O, particularly older patients. Hypoxemia (O2 saturation less than 86%) developed in five patients receiving N2O and in one receiving O2. This difference was not significant. Administration of N2O did not decrease postoperative O2 saturation, nor did it alter the incidence of postoperative hypoxemia, cough, or sputum production.     

Selective elution of HLA antigens and beta 2-microglobulin from human platelets by chloroquine diphosphate

To determine whether chloroquine can specifically elute HLA antigens and beta 2-microglobulin (beta 2-M) from the platelet surface, quantitative immunofluorescence flow cytometry and monoclonal antibodies were used to show that HLA antigens and beta 2-M were proportionally eluted from the platelet surface without affecting the membrane glycoproteins IIb and IIIa. Second, an autoradiogram of electrophoresed I125-labeled platelets showed that only beta 2-M but not other I125-labeled membrane proteins could be eluted. Although HLA antigens were poorly labeled by I125 and could not be detected on the autoradiogram, the eluted HLA antigens could be detected by anti-HLA monoclonal antibody and immunoblotting techniques. No loss of plasma membrane integrity was observed by transmission electron microscopy after chloroquine treatment of platelets. The results indicate that chloroquine selectively elutes HLA antigens and their noncovalently associated beta 2-M without affecting other integral platelet membrane proteins.