Charles W. Putnam

RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast

Eukaryotic checkpoint genes regulate multiple cellular responses to DNA damage. In this report, we examine the roles of budding yeast genes involved in G2/M arrest and tolerance to UV exposure. A current model posits three gene classes: those encoding proteins acting on damaged DNA (e.g. RAD9 and RAD24), those transducing a signal (MEC1, RAD53 and DUN1) or those participating more directly in arrest (PDS1). Here, we define important features of the pathways subserved by those genes. MEC1, which we find is required for both establishment and maintenance of G2/M arrest, mediates this arrest through two parallel pathways. One pathway requires RAD53 and DUN1 (the ‘RAD53 pathway’); the other pathway requires PDS1. Each pathway independently contributes ∼50% to G2/M arrest, effects demonstrable after cdc13‐induced damage or a double‐stranded break inflicted by the HO endonuclease. Similarly, both pathways contribute independently to tolerance of UV irradiation. How the parallel pathways might interact ultimately to achieve arrest is not yet understood, but we do provide evidence that neither the RAD53 nor the PDS1 pathway appears to maintain arrest by inhibiting adaptation. Instead, we think it likely that both pathways contribute to establishing and maintaining arrest.

Dynamic organ culture of precision liver slices for in vitro toxicology

The lack of a reproducible method for the production of thin tissue slices has hindered the use of liver slices as an in vitro tool for hepatotoxicity studies. Fresh human, rat, and rabbit liver was processed using a mechanical slicer. With this instrument, precision (5% of thickness) liver slices in the submillimeter range could be produced at a rapid rate. Slices were prepared from fresh livers in chilled, oxygenated buffer to minimize trauma. Following incubation for up to 20 h in a dynamic organ culture system, histology of incubated slices suggested that 250 m precision-cut slices were optimum in regard to morphology relative to liver slices incubated under conventional organ culture conditions. Addition of bromobenzene to the culture showed time-dependent hepatotoxicity based on two classic parameters of cell degeneration. Histological evidence is presented which suggests the usefulness of this system for hepatotoxicity studies and the production of focal necrosis in vitro.

Portal diversion for the treatment of glycogen storage disease in humans.

Seven patients with Types I, III, or VI glycogen storage disease were treated with portal diversion from 5 1/2 mth to 9 1/2 yr ago. The first 2 patients had portacaval transposition with 1 early death. The last 5 patients had the technically safer procedure of end to side portacaval shunt without any deaths and with no late findings of hepatic encephalopathy. The convalescence of the patients with either kind of portal diversion was characterized by accelerated body growth and bone mineralization, incomplete relief of hypoglycemia and metabolic acidosis, striking amelioration of the hyperlipidemia of Type I disease, liver shrinkage in Types I and VI disease, and variable relief of such diverse other derangements as abnormal bleeding and elevated serum uric acid concentrations. The liver concentrations of glycogen were not affected by portal diversion. Hepatocytes were decreased in size in subsequent biopsies, thereby accounting for the reduction of liver size in most of the cases without a major alteration in glycogen. There was a high incidence of pre existing coincidental liver disease in patients, including transaminase elevations and hepatic fibrosis or even cirrhosis. These abnormalities were particularly striking in Type III patients but did not appear in any of the cases to be made worse by portal diversion. The observations in these 7 patients and in 6 more reported from other centers and followed up with personal examination indicate that portal diversion should have an important role in carefully selected cases of glycogen storage disease. Recent work was reviewed which suggests that the effects of portacaval shunt are due more to the rerouting of pancreatic hormones around the liver than to the bypassing of alimentary glucose.

Prolactin stimulation of ornithine decarboxylase and mitogenesis in Nb2 node lymphoma cells: The role of protein kinase C and calcium mobilization

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination with calcium ionophores has been shown to bypass the requisite antigen- or lectin-induced signal for lymphocyte mitogenesis. This suggests that protein kinase C activation and calcium mobilization may be early events required for lymphocyte proliferation. Therefore, the relationship(s) of protein kinase C activation and calcium mobilization to ornithine decarboxylase induction and cellular proliferation were examined in a rat node lymphoma cell line (Nb2) which is dependent upon prolactin (PRL) for mitogenesis. TPA enhanced PRL-stimulated Nb2 node lymphoma cell ornithine decarboxylase induction and [3H]thymidine incorporation. Addition of a calcium ionophore (A23187) to cultures containing TPA plus PRL increased ornithine decarboxylase above PRL alone or PRL plus TPA but inhibited proliferation compared to the PRL plus TPA regimen. Exposure of cells to TPA or TPA plus A23187 increased [3H]thymidine incorporation in a similar manner to that demonstrated for low-dose PRL. However, optimal concentrations were only 20-25% as effective as mitogens as was optimal PRL. Protein kinase C and calmodulin antagonists inhibited PRL-stimulated ornithine decarboxylase induction and proliferation. Ca2+ chelation or cation channel antagonism inhibited both PRL-stimulated responses. The cyclic AMP analogue, 8Br-cAMP, inhibited PRL-stimulated ornithine decarboxylase activity as well as cellular proliferation processes assessed by [3H]thymidine incorporation. Finally, tumor-promoting phorbol esters inhibited 125I-rPRL binding. These data strongly suggest that protein kinase C activation and calcium mobilization are requisite events for PRL-stimulated ornithine decarboxylase induction and cellular proliferation in Nb2 node lymphoma cells. An additional component that is linked to alterations in K+ channeling is also implicated. These data support a role for protein kinase C in PRL-coupled mitogenesis. However, other critical Ca2+ and/or ion-induced events are also required.

Abdominal wall hernias in patients with abdominal aortic aneurysmal versus aortoiliac occlusive disease

BACKGROUNDnThis study was undertaken to determine the incidence of ventral incisional hernias (VIHs) and inguinal hernias (IHs) in patients with abdominal aortic aneurysmal (AAA) versus those with aortoiliac occlusive disease (AIOD).nnnPATIENTS AND METHODSnThe medical records of 193 patients (128 with AAA and 65 with AIOD) who had undergone elective aortic reconstruction were reviewed to determine the number and location of abdominal wall hernias (AWHs).nnnRESULTSnForty-one AWHs (28 IHs and 13 VIHs) were detected in patients with AAA compared to 13 (11 IHs and 2 VIHs) in patients with AIOD. There was a significantly greater incidence of VIHs in patients with AAA versus patients with AIOD (10% versus 3%, P < 0.05) and recurrent AWHs (28% versus 19%, P < 0.01), but not of IHs (22% versus 17%).nnnCONCLUSIONnPatients with AAA have a higher incidence of VIHs and recurrent AWHs--without a corresponding increase in patient-related risk factors--than patients without aneurysm, suggesting that as yet unidentified etiologic factors may contribute to the development of AWHs in these patients.

Prolactin is a tumor promoter in rat liver

Since prolactin, like the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, induces ornithine decarboxylase and plasminogen activator activities, biochemical markers of a trophic response, this hormone might likewise promote neoplasia. To test this theory, rats were initiated with a hepatocarcinogen followed by six weeks of ovine prolactin. This regimen caused hepatomegaly and the development of enzyme-altered foci. Promotion with prolactin for 23 weeks further increased the numbers of enzyme-altered foci. We suggest that prolactin is an endogenous tumor promoter for chemically initiated cells.

Alopecia, ascites, and incomplete regeneration after 85 to 90 per cent liver resection

A nineteen year old female underwent 85 to 90 per cent partial hepatectomy to treat a minimal deviation hepatoma. Observations afterwards suggested that the limit of resection compatible with survival had been reached. She recovered perfect health after many months, although liver regeneration was not complete. Severe but eventually reversible alopecia and ascites developed postoperatively, undoubtedly as a complication of the massive hepatic resection.

Metabolism of 2,2′,3,3′,6,6′-hexachlorobiphenyl and 2,2′,4,4′,5,5′-hexachlorobiphenyl by human hepatic microsomes☆☆☆

Since the metabolism of polychlorinated biphenyls (PCBs) is the critical factor that determines whether or not they accumulate in adipose tissue, we have studied the metabolism of two hexachlorobiphenyls (HCBs), 2,23,3,6,6-hexachlorobiphenyl (236-HCB) and 2,24,4,5,5-hexachlorobiphenyl (245-HCB), by human hepatic microsomes. Human microsomes were isolated from patients undergoing liver resection and were found to have cytochrome P-450 levels (0.28 nmoles/mg microsomal protein) and cytochrome P-450-dependent enzymatic activities similar to those reported by other workers. 245-HCB was not metabolized by human microsomes under various conditions, while 236-HCB was metabolized with an apparent Km of 8.8 microM and a Vmax of 5.1 pmoles/mg microsomal protein/min. Two major metabolites were formed and identified by gas chromatography-mass spectrometry as 2,2,3,3,6,6-hexachloro-4-biphenylol and 2,2,3,36,6-hexachloro-5-biphenylol. [14C]236-HCB equivalents were found to covalently bind to microsomal protein. Addition of 1 or 5 mM reduced glutathione decreased the degree of covalent binding. These data suggest that HCBs are metabolized through an arene oxide. The fact that 245-HCB was not metabolized explains why it is the predominant PCB found in human adipose tissue.

Hepatic ischemia, caused by celiac axis compression, complicating pancreaticoduodenectomy.

OBJECTIVEnIn the course of pancreaticoduodenectomy, profound hepatic ischemia developed in two patients (one with ampullary carcinoma, the other with chronic pancreatitis). This article addresses the diagnosis and correction of the celiac axis compression responsible in this complication.nnnSUMMARY BACKGROUND DATAnSince hepatic ischemia appeared immediately after division of the gastroduodenal--pancreaticoduodenal arcade, which provides mesenteric to celiac collateral circulation, celiac axis narrowing or occlusion was suspected. Previous reports have indicated that celiac axis disease may be present in about 10% of such patients.nnnMETHODSnDoppler flow studies, and in the second patient, intraoperative angiography were performed. The celiac axis was exposed and mobilized in both.nnnRESULTSnInitially, no flow could be detected in the celiac axis. Dense fibrous tissue was found encasing it. Division of the entrapping tissue restored flow to the upper abdominal viscera.nnnCONCLUSIONSnThe anatomic deformation of the celiac axis predisposing to this complication is detectable on the lateral projection of a preoperative celiac angiogram. If, however, an angiogram has not been done, an initial test occlusion of the gastroduodenal artery before its division permits anticipation of the complication, correction of the celiac impingement, and hence, avoidance of hepatic ischemia.

Biotransformation activity in vitrified human liver slices

In vitro testing of human liver for biotransformation or xenobiotic metabolism studies has been limited by unpredictable acquisition of samples. Consequently, it has become necessary to consider methods to cryopreserve and store these samples whenever they do become available for culture of the revived tissue at a more convenient time. Human liver slices were cryopreserved by vitrification, which allows for the transfer of aqueous media to low temperatures (-196 degrees C) without the formation of ice crystals. Human liver slices were exposed to increasing concentrations of 1,2-propanediol up to a final concentration of 4.76 M in fetal calf serum. Slices were then vitrified by direct immersion into liquid nitrogen and warmed by submersion in 37 degrees C fetal calf serum. Warming was done either immediately or after 4 and 8 weeks of storage under liquid nitrogen. The effects of vitrification, storage time, and warming on biotransformation were determined by assessing the integrated metabolism of 7-ethoxycoumarin (7-EC). Vitrified or fresh human liver slices were exposed to 50 microM 7-EC and its primary metabolite 7-hydroxycoumarin (7-HC) in organ culture for up to 6 hr. Metabolite production of both fresh and vitrified liver slices was compared. Retention of the inherent biotransformation rate was usually high and seemed independent of storage time. Integration of both cytochrome P450-mediated and secondary conjugation processes was retained in cryopreserved tissue. Vitrification offers a way to cryopreserve human liver slices for the study of xenobiotic metabolism in humans.