Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev.

The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.

Technology transfer at US universities: seeking public benefit from the results of basic research.

The development of technology transfer programs at universities throughout the US was catalyzed by the passage of the Bayh-Dole Act in 1980. This landmark legislation allowed universities to own and manage all inventions developed by their employees with federal funding. An effective technology transfer program requires: productive and innovative researchers; clear, supportive policies; knowledgeable professional staff; and adequate funding for operating expenses and for patenting inventions. When all four components are in place, technology transfer can enhance the university's ability to interact with industry and can make a significant contribution to economic development at the local, state and national level.

Extralobar pulmonary sequestration.

A neonate presenting with a density of the right mid-thorax was found, at thoracotomy, to have an extralobar pulmonary sequestration. The sequestration was resected, and the infant made an uneventful recovery. The morphogenesis, diagnosis, and management of this uncommon lesion are discussed.

Ureteropelvic junction obstruction in the newborn.

Ureteropelvic junction (UPJ) obstruction is a common cause of hydronephrosis in infants. Newborns with severe obstruction often have marked improvement following correction; therefore early diagnosis and operation is important. From 1973 to 1983, 21 patients were operated on for UPJ obstruction diagnosed under 6 weeks of age. Six patients (29%) had antenatal ultrasonographic diagnosis. The remaining patients were diagnosed by IVP or radionuclide scan for palpable renal enlargment or for associated anomalies. Seventeen had unilateral and four had bilateral obstruction. Twenty-three pyeloplasties, one primary nephrectomy, and one cutaneous pyelostomy with subsequent nephrectomy were done. All pyeloplasties were dismembered, with tailoring of the renal pelvis. Postoperative renal function was followed with radionuclide scan or IVP. Postoperative complications included a single urinary tract infection in three patients and two bowel obstructions. One early postoperative death occurred in an infant with bilateral obstruction who developed congestive heart failure secondary to severe uncontrollable hypertension. There were two other unrelated late deaths. Documented functional improvement with minimal complications follow unilateral or simultaneous bilateral pyeloplasty in newborns with UPJ obstruction.

A new look at the multifactoral etiology of gallbladder disease in children.

The etiology of gallbladder disease in children is multifactoral. Seven of these factors are fairly well known: obstruction of biliary ductal system, hemolytic disease, estrogen effect, obesity, familial, metabolic, and stress related. Our biggest group of patients, 19 out of the total 61 or 31 per cent, were found to have had prior abdominal/renal surgery or partial bowel obstruction. It has been shown that ileal resections result in gallstones due to loss of bile salt absorption; however, volvulus and partial obstruction from adhesive bands have never been incriminated before. A number of teenagers appear to develop acalculous cholecystitis and have symptoms a good many months before stones develop. These patients who have delayed (greater than 36 hours) excretion of oral cholecystogram dye also have positive duodenal drainage studies after cholecystokinin. Gallbladder disease in children is not a rarity as surgical textbooks would lead one to believe.

Rat liver cholesterol 7 alpha-hydroxylase. Modulation of enzyme activity by changes in phosphorylation state.

Four lines of evidence presented here suggest that the activity of cholesterol 7 alpha-hydroxylase in rat liver is modulated by changes in its phosphorylation state. 1) Livers were homogenized and microsomes were isolated and washed in the presence of either 50 mM NaCl or 50 mM NaF, the latter an inhibitor of phosphoprotein phosphatases. The 7 alpha-hydroxylase activity of microsomes prepared with NaF was 80% greater than that of microsomes prepared with NaCl. 2) Incubation of 10,000 X g supernatants from rat liver for 20 min at 37 degrees C in the absence of 50 mM KF decreased the activity of microsomal cholesterol 7 alpha-hydroxylase by 52%. No significant change was seen in the presence of KF. 3) 7 alpha-Hydroxylase activity fell by 40% when microsomes were incubated with bacterial alkaline phosphatase compared to incubation of microsomes with phosphatase that was inhibited by phosphate and EDTA. 4) 7 alpha-Hydroxylase activity increased by 22% when phosphatase-treated microsomes were incubated for 40 min at 37 degrees C with 1 mM MgATP, 50 microM cAMP, and 200 units of cAMP-dependent protein kinase.

Necrotizing enterocolitis following operation in the neonatal period.

Necrotizing enterocolitis (NEC) usually occurs in low birth weight infants who have had perinatal stress, and the mortality remains significant. There are a few reports of NEC in the postoperative period, especially in young infants. Nine neonates developed NEC following operations and form the basis of this report. The interval between operation and the diagnosis of NEC varied from 3 days to 4 mo. The surgical lesions included one case each of esophageal atresia, tetralogy of Fallot, supralevator rectal atresia with rectourethral fistula, and multiple intestinal atresias. Three babies had gastroschisis and two had "apple peel" intestinal atresia. Only 3 of the 9 survived. The usual clinical findings of NEC, abdominal distention, bile stained gastric residuals and diarrhea (with or without blood), can occur in the postoperative period without NEC and are, therefore, not reliable diagnostic signs. Significant changes in the clinical course of these babies occurred from 7 hr to 5 days before the diagnosis was established. In these patients the roentgen findings that established the diagnosis of NEC included intestinal ileus, pneumatosis intestinalis, and portal vein gas. Pneumatosis intestinalis and portal vein gas were the most reliable diagnostic signs, but appeared relatively late in the course of the disease. In one case pneumatosis was seen only in retrospect. None of the patients had definite pneumoperitoneum. Awareness of NEC as a potential postoperative complication may result in early recognition, treatment and survival.

Tracheal agenesis.

Tracheal agenesis is a rare cause of respiratory distress in the neonatal period. Temporary survival depends on ventilation through the esophagus. Thirty-eight case reports of tracheal agenesis (including one from this institution) have appeared in the literature. In this paper, we present the case reports of our 2 patients and review the literature. Tracheal agenesis is associated with a wide variety of congenital anomalies, the most frequent being ventricular septal defect. A new classification encompassing seven types of tracheal agenesis is described.

Thal fundoplication: a simple and safe operative treatment for gastroesophageal reflux.

One hundred patients with complications of severe gastroesophageal reflux were treated surgically by the Thal fundoplication. In all patients the symptoms of reflux were eliminated by the operation, although 4 recurred within 8 months. Two of these were due to disruption of the fundoplication and two were due to hiatus hernia not recognized and repaired at initial operation. There were 8 deaths, none related to gastroesophageal reflux or the operation. Four patients required re-operation for intestinal obstruction. The Thal fundoplication is a simple procedure which fixes the distal esophagus within the abdomen and produces an acute angle of His. It is effective in prevention of reflux and the patient is able to burp and vomit if necessary. It has not been associated with dysphagia or "gas bloat" which may follow the Nissen fundoplication.

Cyclic AMP-sensitive activation of hepatic sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We previously showed that preincubation of a 10,000 g supernatant (S(10)) from rat liver for 20 min at 37 degrees C dramatically increased the subsequent incorporation of [(14)C]acetate into sterols. No activation was seen with [(14)C]mevalonate as substrate. In the present studies we have examined the effect of preincubation on HMG CoA reductase. When microsomes were isolated from S(10) by calcium precipitation, preincubation of S(10) increased the specific activity of HMG CoA reductase threefold. No activation of HMG CoA reductase was observed in microsomes isolated by ultracentrifugation. Activation was cyclic AMP-sensitive. When cyclic AMP (0.001-1.0 mM) and MgATP (1 mM) were present during the preincubation period, there was little or no activation of HMG CoA reductase activity or of sterol synthesis from acetate. MgATP alone did not prevent activation. Neither cyclic AMP nor MgATP was inhibitory when present only during the assay of sterol synthesis. We propose that the in vitro activation represents the reversal of a physiologic cyclic AMP-mediated mechanism for the control of hepatic HMG CoA reductase. That a phosphoprotein phosphatase may catalyze the activation was supported by the observation that sodium fluoride, an inhibitor of phosphoprotein phosphatases, inhibited the activation. These results suggest that hormone-induced changes in the cellular level of cyclic AMP may regulate the activity of HMG CoA reductase and the rate of hepatic cholesterol synthesis.

Esophageal atresia with double tracheoesophageal fistula.

Ninety cases of esophageal atresia (EA) with double tracheoesophageal fistula (TEF) from the literature and 4 new cases are examined. The incidence may be more common than is generally recognized with incidences up to 5.3% reported. The pre-, intra-, and postoperative diagnosis may be difficult, and half have been missed initially with almost half of these being first recognized at autopsy. During repair of EA and TEF the proximal esophagus should be mobilized looking for a proximal fistula. Many proximal TEF are missed at the initial operation and discovered in the postoperative period. The symptoms, diagnosis and treatment of the unrecognized proximal TEF are similar to that for isolated TEF. Results should be good since this problem occurs in large babies with fewer and less complex associated conditions.

Improved methods for the study of hepatic HMG CoA reductase: one-step isolation of mevalonolactone and rapid preparation of endoplasmic reticulum.

Two new methods are described for the study of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. (1) Endoplasmic reticulum was rapidly prepared by diluting a 10,000 g supernatant with buffer containing 8 mM calcium chloride. The yield of protein and the specific activity of HMG CoA reductase in the pellet subsequently obtained by low speed centrifugation were nearly identical to those in the microsomal pellet prepared by ultracentrifugation. This technique may be particularly useful in studies of the rapid, in vitro modulation of the enzyme. (2) Mevalonolactone was extracted into benzene from the HMG CoA reductase assay mixture with an efficiency of 58%. There was less than 1% extraction of HMG CoA, acetoacetate, or beta-hydroxybutyrate. The extracted mevalonolactone was at least 98% pure as judged by thin-layer chromatography with four different solvent systems. These improved methods should significantly aid studies of the physiological importance of HMG CoA reductase.