Hierarchic organization of globular proteins: Experimental studies on thermolysin

Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316 and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary structure of the fragments show cooperativity with a temperature of half-denaturationT _mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316 of thermolysin.     

The effects of aluminum loading on the renal response to parathyroid hormone in the vitamin D-replete rat

Aluminum (Al) may cause vitamin D-resistant osteomalacia and depress the serum levels of immunoreactive parathyroid hormone (iPTH) in patients treated with maintenance dialysis and those on total parental nutrition (TPN). Both conditions have been associated with low serum levels of 1,25(OH)2-vitamin D (1,25(OH)2D). Al may inhibit PTH secretion in vitro; however, induction of hypocalcemia can enhance endogenous PTH secretion in Al-loaded dogs and TPN patients. Despite hypocalcemia and/or increased endogenous iPTH levels, Al-loaded TPN patients fail to show the expected rise in serum 1,25(OH)2D levels. Such observations suggest that Al may impair the renal response to PTH. We studied vitamin D-replete rats given Al or saline vehicle IP for 5 days. Al and control rats then received a saline infusion with an IV bolus of PTH 1-34. Urinary cyclic AMP and P excretion rose in Al and control rats by 1 hr post-PTH, without differences between the groups. Serum P and ionized Ca levels were not different between Al and control rats. In other Al and control rats, serum 1,25(OH)2D levels were measured after saline without PTH. Serum 1,25(OH)2D levels were higher in controls given PTH than in those without, but 1,25(OH)2D levels were not different between Al rats given PTH and those with none. Thus, aluminum does not affect cyclic AMP or P excretion but may impair 25(OH)D-1 alpha-hydroxylase activity in response to PTH.     

Correlation of an estrogen receptor-related phosphoprotein with histopathological features in breast cancer

A series of 65 cases of different histological types of breast carcinoma was investigated for the immunohistochemical location of the estrogen receptor-related, 29 kD phosphoprotein using the ER-D5 monoclonal antibody. The ER-D5 response is heterogeneous in relation to some therapeutic limitations and is correlated with histopathological features of the tumors and survival. The main parameters for evaluation of breast cancers are reviewed, both those that are statistically correlated and those that are not apparently always correlated but are known to have considerable biological meaning, such as the ER-status of tumors.     

Kinetics of tert-[35S]Butylbicyclophosphorothionate Binding in the Cerebral Cortex of Newborn and Adult Rats: Effects of GABA and Receptor Desensitization

Abstract: The effects of GABA on the kinetics of tert -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the convulsant site of GABA_A receptors were studied in membrane suspensions from the cerebral cortex of newborn (1-day-old) and adult (90-day-old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [³⁵S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast ( t _1/2, 11 min) and slow ( t _1/2, 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 µ M GABA, however, was more robust in neonates than in adults (six- to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 µ M GABA (dissociation started by adding 40 µ M picrotoxin) were two- to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 µ M picrotoxin plus 2 µ M GABA). Taken together, these results suggest that (1) the closed state of GABA_A receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS.     

Fungal biodiversity in the air of Turin

The qualitative fungal composition of Turin 's atmospheric environment was surveyed, carrying out a twelve-month study and collecting with a single stage volumetric sieve sampler on Dermasel agar supplemented with 0.4 g l^–1 cycloheximide and 0.05 g l^–1 chloramphenicol. We isolated 165 species and 2 varieties of mesophilic fungi from 58 genera and 26 thermotolerant species from 12 genera. Penicillium, Aspergillus, Acremonium, Chrysosporium, Scopulariopsis, Malbranchea, Paecilomyces, Phialophora and Cladosporium were in sequence the genera most rich in mesophilic species; Aspergillus, Penicillium, Chrysosporium and Scopulariopsis the most rich in thermotolerant species. Many of the species isolated are rarely or never recorded in the atmospheric environment. Cycloheximide can thus be said to select among airborne fungi, giving a characteristic picture.Abbreviation CH Cycloheximide - cfu colony forming unit     

Modulation of drug-induced cytotoxicity by a bispecific monoclonal antibody that recognizes the epidermal growth factor receptor and doxorubicin

A hybrid hybridoma secreting a bispecific hybrid mAb (bsmAb), which recognizes both the epidermal growth factor receptor (EGF-R) and the drug doxorubicin, was produced by somatic hybridization of two hybridomas. The bsmAb obtained was able to retarget doxorubicin cytotoxicity in vitro specifically on EGF-R-positive cells exerting at the same time an antidotal effect on cells that did not overexpress the EGF-R. Distribution studies in mice indicate that the bsmAb selectively delivers the drug to tumour cells and modifies doxorubicin biodistribution with a statistically significant decrease of drug concentration in the intestine, which is the main target of early anthracycline toxicity. In keeping with this finding is the remarkable antidotal activity exerted by bsmAb in mice treated with doxorubiein, which is proved by retardation in loss of body weight and mortality. The effectiveness on tumour growth of the mAb followed by the administration of doxorubicin appears to be equal to that of the drug alone; however, the bsmAb exerts a remarkable antidotal activity.     

H1° and H3.3B mRNA levels in developing rat brain

Two overlapping rat cDNAs, covering a continuous region of 1107 base pairs, have been isolated and sequenced. The clones contain identical open reading frames, encoding a 136 amino acid long polypeptide which exhibits 100% identity to other mammalian H3.3 histone variants. We show that the inserts derive, in particular, from the H3.3B gene. We used these inserts and an insert from an H1° encoding clone, previously described (6), as probes to study the accumulation of mRNAs encoding the corresponding histone replacement variants (namely, H1° and H3.3) during rat brain development. We found that the concentration of both H1° and H3.3B mRNAs decreases from the embryonal day 18 (E18) to the postnatal day 10 (P10), with inverse correlation to protein accumulation.This paper is dedicated to our friend Paolo Carbone who devoted his life to research and teaching in Genetics. We will always remember him for scientific honesty and for his unique qualities of humanity.