Identification of cysteine pairs within the amino-terminal 5% of apolipoprotein B essential for hepatic lipoprotein assembly and secretion

There is growing evidence that the amino-terminal globular domain of apolipoprotein B (apoB) is essential for lipoprotein particle formation in the hepatic endoplasmic reticulum. To identify the structural requirements for its function in lipoprotein assembly, cysteine (Cys) pairs required to form the seven disulfide bonds within the amino-terminal 21% of apoB were replaced in groups or individually by serine. Substitution of Cys pairs required for formation of disulfide bonds 1-3 or 4-7 (numbered from amino to carboxyl terminus) completely blocked the secretion of apoB28 in transfected HepG2 cells. To identify the specific disulfide bonds required for secretion, Cys pairs were mutated individually. Substitution of Cys pairs required for disulfide bonds 1, 3, 5, 6, or 7 had little or no impact on apoB28 secretion or buoyant density. In contrast, individual substitution of Cys pair 2 (amino acid residues 51 and 70) or 4 (218 and 234) severely inhibited apoB28 secretion and its capacity to undergo intracellular assembly with lipid. The same assembly and secretion defects were observed when these mutations were expressed as part of apoB50. These studies provide direct evidence that the ability of the internal lipophilic regions of apoB to engage in the recruitment and sequestration of lipid during translation is critically dependent upon a structural configuration contained within or affected by the amino-terminal 5% of the protein.     

Late onset peripheral seronegative spondyloarthropathy: report of two additional cases

Two more cases of late onset peripheral seronegative spondyloarthropathy (SpA) are reported. Like the patients reported by Dubost and Sauvezie, they had extensive pitting edema of the lower limbs, constitutional symptoms, elevated erythrocyte sedimentation rate and minimal involvement of the axial skeleton with marked signs of diffuse idiopathic skeletal hyperstosis.     

Virus load as a marker of disease progression in HIV-infected children

The relationship of virus load to clinical disease progression in HIV-infected children remains to be elucidated. In this study, HIV-1 proviral DNA load was determined in peripheral blood mononuclear cells (PBMCs) by the quantitative competitive DNA polymerase chain reaction assay (QC-PCR) in 47 HIV-infected children subdivided by age (group I, < or = 2 years; group II, > or = 5 years), who were further categorized to include 12 rapid progressors (RP, age < or = 2 years, Centers for Disease Control [CDC] defined clinical category C and/or immune category 3, or death before age 2 years) and slow progressors (SP, age > or = 5 years, excluding CDC categories C and/or immune category 3). Significantly higher mean proviral copies/10(3) PBMCs were detected in group I versus group II (75.4 +/- 104.3 and 13.0 +/- 17.8 respectively, p < 0.0001) and in RP (158.0 +/- 118.2) as compared to either SP (11.8 +/- 18.8, p < 0.0001) or other age-matched infected children (20.3 +/- 38.8, p < 0.0001). Thus HIV-infected children appear to have a higher cell-associated virus load early in life, especially in association with rapid disease progression.     

Inhalation toxicity of 1,6-hexanediamine dihydrochloride in F344/N rats and B6C3F1 mice

1,6-Hexanediamine (HDA) is a high production volume chemical which is used as an intermediate in the synthesis of paints, resins, inks, and textiles and as a corrosion inhibitor in lubricants. Two- and 13-week studies of the toxicity of the dihydrochloride salt of HDA (HDDC) were conducted in male and female Fischer 344/N rats and B6C3F1 mice using whole-body inhalation exposure. Both species were evaluated for histopathologic and reproductive effects, and rats were examined for clinical chemistry and hematologic changes. In the 2-week inhalation studies, animals were exposed to 10-800 mg HDDC/m3, 6 hr per day. All rats, all female mice, and two of five male mice in the high-exposure group died before the end of the study. Surviving mice in this group had a dose-dependent depression in body weight gain. Clinical signs were primarily related to upper respiratory tract irritation and included dyspnea and nasal discharge in both species. Treatment-related histopathologic lesions included inflammation and necrosis of the laryngeal epithelium of both species and the tracheal epithelium of mice, as well as focal inflammation and ulceration of the respiratory and olfactory nasal mucosa. In the 13-week inhalation studies, animals were exposed to HDDC at concentrations of 1.6-160 mg/m3 for 6 hr per day, 5 days per week. In addition to the base study groups, a supplemental group of rats at each exposure level was included to assess the effect of HDDC on reproduction. No treatment-related changes in organ weights or organ-to-body-weight ratios occurred in rats, and no treatment-related clinical signs or gross lesions were seen in either species. Chemical-related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) in the two highest exposure groups and were similar in both species. These lesions included minimal to mild focal erosion, ulceration, inflammation, and hyperplasia of the laryngeal epithelium, in addition to degeneration of the olfactory and respiratory nasal epithelium. HDDC caused no significant changes in sperm morphology or vaginal cytology and no significant adverse effects on reproduction in rats or mice. Hematologic and clinical chemistry changes in rats were minor and sporadic and were not accompanied by related histologic findings. HDDC did not increase the frequency of micronucleated erythrocytes in mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methods to compare dissolution profiles and a rationale for wide dissolution specifications for metoprolol tartrate tablets

The objectives of this work were to apply several profile comparison approaches to dissolution data of four different but bioequivalent metoprolol tartrate tablet formulations to (1) identify the advantages and disadvantages of each approach, (2) quantify the metric for comparing dissolution profiles of each method, (3) determine metric limits that are consistent with the observed bioequivalence, and (4) rationalize the observed metric limits with respect to the role of dissolution in overall metoprolol absorption. Dissolution was performed by the USP monograph method on four formulations of metoprolol tartrate tablets (Lopressor plus fast, medium, and slow dissolving test formulations). Three general approaches to compare dissolution profiles were examined; they were ANOVA-based, model-independent, and model-dependent approaches. It is concluded that model-independent approaches and several model-dependent approaches yielded numerical results that can serve as objective and quantitative metrics for comparing entire dissolution profiles of the four metoprolol tartrate formulations. However, these methods presented complications. Some metrics were dependent on the length of the dissolution profile and the sampling scheme. Results from the pairwise procedures also depended on the pairing assignment of individual profiles. In spite of complications, these methods suggested wide dissolution specification limits. Wide dissolution specifications were rationalized through an analysis of in vitro-in vivo relationships, which indicated metoprolol dissolution from these formulations was not the rate-limiting step; hence, a range of dissolution profiles can be expected to yield equivalent plasma profiles.     

Bovine ovarian non-genomic progesterone binding sites: presence in follicular and luteal cell membranes

We have shown recently that the bovine corpus luteum (CL) possesses specific luteal cell surface membrane binding sites for progesterone. We have now confirmed and extended these observations to compare the subcellular distribution of these binding sites in developing, mature and regressing CL. The median buoyant densities of luteal progesterone binding sites from early-, mid- and late-luteal phase CL were similar (though three of five density profiles for late-luteal phase CL showed association of steroid binding with a fraction with a lower density), and clearly resolved from nuclear, mitochondrial, lysosomal, peroxisomal, Golgi-endoplasmic reticulum-lysosomal and smooth endoplasmic reticulum markers. Specific binding of [3H]progesterone overlapped with the distributions of 5'-nucleotidase and luteinizing hormone receptor (luteal cell surface membrane markers) in both control and digitonin-treated gradients at all stages of the luteal phase. Since steroidogenic 'large luteal' and 'small luteal' cells of the CL are derived from the granulosa cells (GC) and theca of the preovulatory follicle, we also investigated whether similar receptors were present in the follicle, and describe for the first time specific membrane binding sites for progesterone in purified GC and thecal membranes from healthy bovine follicles of different sizes. Specific binding increased linearly with GC and thecal membrane protein concentration; however, it was detectable only when digitonin was included in the binding incubation. Binding sites were specific for progesterone; unlabelled progesterone competed for [3H]progesterone binding at low concentrations (IC50, 35 and 31 nmol/l) compared with testosterone (IC50, 905 and 870 nmol/l) and delta4-androstenedione (IC50, 1050 and 660 nmol/l) for GC and thecal receptors respectively. In contrast, oestradiol, oestrone, pregnenolone, cortisol, cholesterol, and a genomic progesterone receptor antagonist, RU486, competed poorly. Steroid binding was present in GC and thecal membranes of follicles of all sizes, but [3H]progesterone binding to GC membranes decreased significantly with increasing follicle size (P<0.02), perhaps indicating developmental regulation of GC membrane non-genomic progesterone receptors in the preovulatory bovine follicle. We suggest that these membrane steroid receptors may be involved in the autocrine/paracrine regulation of follicular function by progesterone.     

Inhaled induction and emergence from desflurane anesthesia in the ambulatory surgical patient: the effect of premedication

We studied the effect of premedication (1 microgram/kg fentanyl and 0.04 mg/kg midazolam 5 min before induction of anesthesia) on airway reactivity and hemodynamic stability during inhaled induction using desflurane in 10 ambulatory surgical patients. Eight patients who were anesthetized without premedication served as the controls. Induction and emergence were rapid and unaffected by premedication. End-tidal and inspired concentrations of desflurane at loss of consciousness were significantly reduced by premedication (10.1% end-tidal/14.1% inspired, no premedication, vs. 5.3% end-tidal/8.9% inspired, premedication). Airway irritability was markedly attenuated by premedication (100% no premedication versus 30% premedicated), as was apnea (37.5% no premedication versus 0% premedicated). We observed an increase in mean arterial blood pressure and heart rate after loss of consciousness (mean arterial pressure 103 vs 121 mm Hg, heart rate 73 vs 100 bpm) in the unpremedicated patients, whereas both groups demonstrated a decrease in mean arterial blood pressure with no change in heart rate when baseline values were compared to those at incision (103 vs 74 mm Hg, no premedication, 99 vs 81 mm Hg premedicated). Patient acceptability was satisfactory and unchanged by premedication. We recommend the use of such premedication when desflurane is used during the induction of anesthesia.