Inositols in treating polycystic ovary syndrome and non-insulin dependent diabetes mellitus: now and the future.

INTRODUCTION: This Expert Opinion covers recent updates in the use of Inositol in polycystic ovary syndrome (PCOS) and type II diabetes and gives support to researchers and clinicians. AREAS COVERED: This article discusses the role of Myo-Inositol (MI) and D-Chiro-Inositol (DCI) in physiological function, the use of MI in PCOS, the risks of using DCI in reproductive conditions, the 40:1 combination of MI/DCI in PCOS. Furthermore, we discuss the issues of insulin resistance and how α-lactalbumin may increase the intestinal bioavailability of MI. The paper then transitions to talk about the use of inositols in diabetes, including type II diabetes, Gestational Diabetes Mellitus (GDM), and double diabetes. Literature searches were performed with the use of PubMed, Google Scholar, and Web of Science between July and October 2023. EXPERT OPINION: Inositol therapy has grown in the clinical field of PCOS, with it demonstrating an efficacy like that of metformin. The use of α-lactalbumin has further supported the use of MI, as issues with intestinal bioavailability have been largely overcome. In contrast, the effect of inositol treatment on the different PCOS phenotypes remains an outstanding question. The use of inositols in type II diabetes requires further study despite promising analogous data from GDM.

Bovine-derived α-lactalbumin exhibits cardiovascular protection against aging by ameliorating the inflammatory process in mice.

Cardiovascular aging is the most important factor leading to cardiovascular disease (CVD), and the incidence and severity of cardiovascular events increase with age. Cardiovascular disease is one of the leading causes of death in the aging population. Therefore, it is extremely urgent to develop and explore effective drugs or bioactive molecules to prevent cardiovascular aging and related diseases. In the current work, the effect of bovine α-lactalbumin (α-lactalbumin is one of the major bioactive protein molecules in milk) on cardiovascular aging was investigated in vitro and in vivo. First, a cellular model of cardiovascular aging was established using H2O2-induced in vitro cellular models. It was found that α-lactalbumin could alleviate cardiovascular senescence by assessing Sa-ß-gal and senescence-related markers (such as p16/p21/p53) in in vitro cellular models. Bovine α-lactalbumin attenuated aging-related inflammation and oxidative stress. Furthermore, aged mice were used as an in vivo cardiovascular aging model. We explored the effect of α-lactalbumin on cardiovascular aging and found that cardiovascular aging was significantly attenuated by evaluating Sa-ß-gal staining and aging-related marker molecules. Mechanistically, we found that α-lactalbumin may alleviate cardiovascular aging by regulating the expression of Sirt1 (Sirtuin 1). In summary, in the current work, we systematically explored the potential biological activity of α-lactalbumin against cardiovascular aging and found that α-lactalbumin has good anti-aging potential in vitro and in vivo, suggesting that α-lactalbumin could be used as an antiaging functional food in the future.

Positive Effects of α-Lactalbumin in the Management of Symptoms of Polycystic Ovary Syndrome.

To date, the involvement of α-Lactalbumin (α-LA) in the management of polycystic ovary syndrome (PCOS) refers to its ability to improve intestinal absorption of natural molecules like inositols, overcoming the inositol resistance. However, due to its own aminoacidic building blocks, α-LA is involved in various biological processes that can open new additional applications. A great portion of women with PCOS exhibit gastrointestinal dysbiosis, which is in turn one of the triggering mechanisms of the syndrome. Due to its prebiotic effect, α-LA can recover dysbiosis, also improving the insulin resistance, obesity and intestinal inflammation frequently associated with PCOS. Further observations suggest that altered gut microbiota negatively influence mental wellbeing. Depressive mood and low serotonin levels are indeed common features of women with PCOS. Thanks to its content of tryptophan, which is the precursor of serotonin, and considering the strict link between gut and brain, using α-LA contributes to preserving mental well-being by maintaining high levels of serotonin. In addition, considering women with PCOS seeking pregnancy, both altered microbiota and serotonin levels can induce later consequences in the offspring. Therefore, a deeper knowledge of potential applications of α-LA is required to transition to preclinical and clinical studies extending its therapeutic advantages in PCOS.

SIRT1 Mediates Neuroprotective and Neurorescue Effects of Camel α-Lactalbumin and Oleic Acid Complex on Rotenone-Induced Parkinson's Disease.

Parkinson's disease (PD) is the second most common devastating neurodegenerative disorder. Presently used therapies for PD have severe side effects and are limited to only temporary improvement. Therefore, a new therapeutic approach to treat PD urgently needs to be developed. α-Lactalbumin, the most abundant milk protein in camel milk, has been attributed to various medicinal properties. This study intended to investigate the neuroprotective efficacy of the camel α-lactalbumin and oleic acid (CLOA) complex. One mechanism postulated to underlie neuroprotection by the CLOA complex is the induction of silent information regulatory protein (SIRT1). SIRT1 is known to be involved in several pathological and physiological processes, and it has been suggested that SIRT1 plays a protective role in PD. Oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis are involved in PD pathogenesis. Our results revealed that SIRT1 inhibits oxidative stress by maintaining HIF-1α in a deacetylated state. SIRT1 upregulates the expression of FOXO3a and HSF-1, thus inhibiting apoptosis and maintaining the homeostasis of cellular proteins. Increased SIRT1 expression reduces the levels of TNF-α, IL-6, and IL-8, which in turn inhibits neuroinflammation. In addition to SIRT1, the CLOA complex also enhances the expression of survivin and leptin and promotes the survival of neuroblastoma cells. Altogether, our results suggest that the CLOA complex might be a novel therapeutic molecule that could ameliorate neuronal cell damage in PD.

A multicenter clinical study with myo-inositol and alpha-lactalbumin in Mexican and Italian PCOS patients.

OBJECTIVE: This open-label non-randomized clinical study aimed at evaluating the effects of myo-inositol plus alpha-lactalbumin in two groups of PCOS women, treated in Mexico and Italy. Alpha-lactalbumin was used being effective in increasing myo-inositol intestinal absorption. This effect is very useful in greatly reducing the therapeutic failure of myo-inositol in some patients (inositol resistant subjects). PATIENTS AND METHODS: The study involved 34 normal weight or overweight patients (14 in Mexico and 20 in Italy), aged 18 to 40 years, with anovulation and infertility > 1 year and insulin resistance diagnosed by HOMA-Index. Patients were administered orally with 2 g myo-inositol, 50 mg alpha-lactalbumin, and 200 µg of folic acid twice a day for 6 months. Controls were the same patients at t0 (baseline). The primary outcome was HOMA-index decrease after 3 and 6 months of treatment. Other parameters monitored were BMI, progesterone, LH, FSH, total testosterone, free testosterone, androstenedione, total cholesterol, HDL, LDL, triglycerides. RESULTS: Recovery was general, and its relevance was higher when the starting point was further away from the normal range. The most important results were obtained with insulin, HOMA-index, LH, and androstenedione. No significant adverse effects were detected in both groups of patients. CONCLUSIONS: This clinical trial demonstrated for the first time that myo-inositol and alpha-lactalbumin improve important parameters in PCOS patients characterized by different metabolic profiles.

Investigation and comparison of the protective activities of three functional proteins-lactoferrin, α-lactalbumin, and ß-lactoglobulin-in cerebral ischemia reperfusion injury.

The objective of this study was to evaluate the protection conferred by lactoferrin, α-lactalbumin, and ß-lactoglobulin in cerebral ischemia reperfusion (I/R) injury. Rat pheochromocytoma (PC12) cells were used to construct an oxygen and glucose deprivation model in vitro, and ICR mice underwent carotid artery "ligation-relaxation" to construct a cerebral I/R injury model in vivo. The levels of toll-like receptor 4 (TLR4) and downstream factors including nuclear factor-κB, tumor necrosis factor-α, and IL-1ß were measured. Metabonomics detection and data mining were conducted to identify the specific metabolic sponsor of the 3 proteins. The results showed that lactoferrin, α-lactalbumin, and ß-lactoglobulin protected neurons from cerebral I/R injury by increasing the level of bopindolol and subsequently inhibiting the TLR4-related pathway to different degrees; ß-lactoglobulin had the strongest activity of the 3 proteins. In summary, this study is the first to investigate and compare the protective effects of lactoferrin, α-lactalbumin, and ß-lactoglobulin in a cerebral stroke model. The results implicate TLR4 as a novel target of the 3 bioactive proteins to prevent cerebral I/R injury.

Dietary α-Lactalbumin protects against thioacetamide-induced liver cirrhosis by maintaining gut-liver axis function in rats.

We tested the hypothesis that α-lactalbumin inhibits the disruption of intestinal barrier function and liver cirrhosis by restoring gut-liver axis function in thioacetamide (TAA) -treated rats. Rat diets were supplemented with α-lactalbumin replacing 50% of dietary protein. After consuming α-lactalbumin for one week, rats were intraperitoneally injected with TAA twice a week for 14 weeks. The α-lactalbumin-enriched diet significantly inhibited the elevation of plasma alanine aminotransferase, aspartate aminotransferase, and hyaluronic acids. The supplement significantly reduced plasma lipopolysaccharide levels and increased occludin mRNA level. Hepatic fibrosis and regenerative nodules was developed and intestinal villi were shortened by TAA; α-Lactalbumin attenuated these histopathological changes. These results indicated that α-lactalbumin improved intestinal barrier function, suppressing endotoxin levels. These data also suggested that α-lactalbumin ameliorated the impairment of the gut-liver axis by TAA, inhibiting the development of liver cirrhosis.

Effects of micronised dispersible ferric pyrophosphate combined with alpha-lactalbumin in pregnant women affected by iron deficiency anemia: results from a prospective, double-blind, randomized controlled trial.

OBJECTIVE: This study aimed at evaluating the effects obtained by administering 30 mg of micronised dispersible ferric pyrophosphate plus 300 mg of alpha-lactalbumin (MDFP-AL) compared to 80 mg of ferrous gluconate (FG) in pregnant women affected by iron-deficiency anemia (IDA). PATIENTS AND METHODS: We considered eligible all second-trimester singleton pregnancies in women affected by IDA. We excluded any other disease, twin pregnancies, any other pharmacologic/nutraceutical treatments (besides folic acid) before/during pregnancy. We randomized patients in two groups: one underwent treatment with 1 tablet of MDFP-AL/day, the other one with 1 tablet of FG/day, for 30 days. We evaluated hemoglobin (Hb), ferritin, red blood cells (RBCs), serum iron, hematocrit (Hct), and side effects at baseline (T0), after 15 days (T1) and 30 days (T2). RESULTS: 50 women met the inclusion/exclusion criteria. We did not observe significant differences between the two groups for mean age, gestational age at the enrollment and parity. In MDFP-AL group, after 15 days (T1) Hb, ferritin, serum iron and Hct and were significantly improved respect to baseline (T0); after 30 days (T2), all the parameters, including RBCs, were significantly improved respect to baseline (T0). Similarly, in FG group the investigated parameters were improved both after 15 (T1) and 30 days (T2) respect to baseline (T0), although less in percentage terms respect to MDFP-AL group. The side effects rate was 24% in FG group, whereas MDFP-AL group did not show any significant side effect. CONCLUSIONS: Overall, MDFP-AL is more effective and safe than FG for the treatment of IDA in pregnant women.

Bovine milk-derived α-lactalbumin inhibits colon inflammation and carcinogenesis in azoxymethane and dextran sodium sulfate-treated mice.

Cyclooxygenase-2 is expressed early in colon carcinogenesis and plays crucial role in the progress of the disease. Recently, we found that α-lactalbumin had anti-inflammatory activity by inhibiting cyclooxygenase-2. In experiment 1, we investigated the effects of α-lactalbumin on the colon carcinogenesis initiated with azoxymethane (AOM) followed by promotion with dextran sodium sulfate (DSS) in mice. Dietary treatment with α-lactalbumin decreased fecal occult blood score at 3 days after DSS intake. α-Lactalbumin also decreased the colon tumor at week 9. In experiment 2, AOM-treated mice were sacrificed at 7 days after DSS intake. The plasma and colon prostaglandin E2 (PGE2) levels in AOM/DSS-treated mice were higher than those in the DSS-treated mice without initiation by AOM. α-Lactalbumin decreased PGE2 in both plasma and colon. These results suggest that α-lactalbumin effectively inhibited colon carcinogenesis, and the inhibition may be due to the decreased PGE2 by inhibiting cyclooxygenase-2 at cancer promotion stages.

Chronic administration of bovine milk-derived α-lactalbumin improves glucose tolerance via enhancement of adiponectin in Goto-Kakizaki rats with type 2 diabetes.

Inflammation has been implicated in development of the insulin resistance that leads to elevated blood sugar levels associated with type 2 diabetes. It is reported that salsalate, a common non-steroidal anti-inflammatory drug, has been shown to decrease blood glucose concentration in some clinical study. Recently, we found that bovine milk-derived α-lactalbumin had anti-inflammatory activity caused by inhibiting cyclooxygenase-2. In this study, we investigated the effects of chronic administration of α-lactalbumin on glucose tolerance in Goto-Kakizaki (GK) rats, a model of type 2 diabetes. After 10 weeks administration of the α-lactalbumin (300 mg/kg, twice a day), oral glucose tolerance tests revealed significant decrements of blood glucose levels after glucose loading. However, significant differences of insulin levels were not observed among three GK rats groups after glucose loading. α-Lactalbumin treatment enhanced high molecular weight form of adiponectin and suppressed prostaglandin E2 levels in plasma. These results suggest that α-lactalbumin effectively decreased blood glucose levels after glucose loading in GK rat, and the decrements may be due to enhancement of adiponectin.

Effects of daily intake of yoghurt enriched with bioactive components on chronic stress responses: a double-blinded randomized controlled trial.

Chronic stress has a negative influence on health. The aim was to determine stress reducing effects of yoghurt enriched with bioactive components as compared to normal yoghurt. High-trait anxiety individuals (n = 67) aged 18-63 years participated in a randomized, placebo-controlled, double-blinded intervention with parallel groups. They received either yoghurt enriched with alpha-lactalbumin, casein tripeptides and B vitamins (active) or isoenergetic standard yoghurt (control). To detect changes in psychological and physiological stress, State-Trait Anxiety Inventory, Profile of Mood States, salivary cortisol, inflammatory markers, blood pressure, heart rate variability (HRV) and actigraphy were monitored. We observed higher ratings of vigor (p = 0.047) and reduced feeling of inefficiency (p = 0.048) in the active group. HRV (baseline adjusted mean 49.1 ± 2.3 ms) and recovery index (106.6 ± 33.4) were higher in the active group than in controls (42.5 ± 2.2 ms and 80.0 ± 29.3) (p = 0.046 and p = 0.02, respectively). In conclusion, daily intake of yoghurt enriched with bioactive components may aid in stress coping.

Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells).

BACKGROUND: Most colon cancers start with dysregulated Wnt/ß-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer treatment is logical, based on the properties of the complex and its biological context. OBJECTIVE: To investigate if HAMLET can be used for colon cancer treatment and prevention. Apc(Min)(/+) mice, which carry mutations relevant to hereditary and sporadic human colorectal tumours, were used as a model for human disease. METHOD: HAMLET was given perorally in therapeutic and prophylactic regimens. Tumour burden and animal survival of HAMLET-treated and sham-fed mice were compared. Tissue analysis focused on Wnt/ß-catenin signalling, proliferation markers and gene expression, using microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells. RESULTS: Peroral HAMLET administration reduced tumour progression and mortality in Apc(Min)(/+) mice. HAMLET accumulated specifically in tumour tissue, reduced ß-catenin and related tumour markers. Gene expression analysis detected inhibition of Wnt signalling and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered ß-catenin integrity and localisation through an ion channel-dependent pathway, defining a new mechanism for controlling ß-catenin signalling. Remarkably, supplying HAMLET to the drinking water from the time of weaning also significantly prevented tumour development. CONCLUSIONS: These data identify HAMLET as a new, peroral agent for colon cancer prevention and treatment, especially needed in people carrying APC mutations, where colon cancer remains a leading cause of death.

The effects of native whey and α-lactalbumin on the social and individual behaviour of C57BL/6J mice.

Milk proteins are the main components of everyday feeding and demonstrate a promising potential to change the mental condition. However, the effects of milk proteins after prolonged use remain poorly understood. The aim of the present study was to compare the effects of two whey proteins (alpha-lactalbumin (alpha-lac) and native whey) with casein on social and individual behaviour in mice. During a 30 d-long dietary intervention, male C57BL/6J mice had ad libitum access to an experimental diet containing 17% (w/w) of one of three protein sources: a-lac, native whey or casein. Mice had voluntary access to a running wheel. Social behaviour (group and resident-intruder activity) was tested at baseline and at the end of the intervention. Half of each dietary group was then withdrawn from the diet and running wheel for 7 d, and social activity and individual behaviour tests (open field, elevated-plus maze, light­dark box and forced swimming) were performed, to evaluate anxiety and depression-like status. The study shows that the long-term ingestion of whey proteins may modulate behaviour when compared with casein. Diet enriched with a-lac exhibited anxiolytic and antidepressive activities while the whey diet improved sociability. The differences between the diet groups were pronounced under the running wheel and the withdrawal of the experimental diet, suggesting that the beneficial effects of the milk proteins are clearer in stressful situations. Diet-induced behavioural changes remained visible for a week after feeding, which suggests that the proteins of the milk whey fraction have prolonged efficacy on the mental state of mice.

A molecular complex of bovine milk protein and oleic acid selectively kills cancer cells in vitro and inhibits tumour growth in an orthotopic rat bladder tumour model.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Novel intravesical therapies are needed for superficial bladder cancer that reduce the risk of infection associated with Bacillus Calmette-Guérin (BCG) and further destabilization of the urothelium associated with cytotoxic chemotherapy. Experimental therapies to date have included photodynamic therapy, oncolytic viruses, gene therapy (antisense oligonucleotides and silencing RNA), cytokine therapy, death receptor agonists (tumour-necrosis-factor-related apoptosis-inducing ligand and anti-DR5 monoclonal antibody), naturally occurring substances (curcumin and deguelin) and human α-lactalbumin made lethal to tumour cells (HAMLET). HAMLET, a natural occurring product in milk, induces apoptosis in urothelial cancer cells but has limitations in clinical application because of its human source. A previous study in patients with bladder cancer has demonstrated that intravesical HAMLET (daily for 5 days before tumour resection) caused selective apoptotic tumour cell death. BAMLET, the bovine equivalent of HAMLET, is a complex of bovine α-lactalbumin and oleic acid (bLAC) that has been shown in vitro to accumulate in the endolysosomal compartment of tumour cells and induce leakage of lysosomal cathepsins into the cytosol followed by activation of the pro-apoptotic protein Bax. This is the first in vivo study to show that BAMLET (bLAC) induces apoptosis in urothelial cancer cells and controls the growth of high risk urothelial cancer in a syngeneic rat orthotopic model. This same bladder cancer model system has been used to test other novel therapies, including BCG, and therefore provides a relative comparison of its effectiveness with other intravesical therapies. OBJECTIVE: To investigate the efficacy of a complex of bovine α-lactalbumin and oleic acid (bLAC) to kill urothelial cancer cells in vitro and inhibit tumour growth and progression in a high risk bladder tumour model. MATERIALS AND METHODS: The cytotoxicity of bLAC to a large panel of urothelial cell cancer (UCC) cells was tested by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay, using bLA, the folded α-lactalbumin without oleic acid, as a control. The mechanism of bLAC-inducing cell death was evaluated by annexin V staining, TUNEL (terminal deoxynucleotidyl transferase mediated nick end labelling) assay and sub-G1 DNA analysis. The selective bLAC cytotoxicity was examined using multicellular spheroids consisting of UCC and non-transformed fibroblasts. Rats bearing orthotopic tumour received intravesical instillations (twice weekly, for 3 weeks) of bLAC, bLA, BCG or saline, starting 6 days after UCC (AY-27) cell inoculation. Animals were monitored for survival, toxicity and tumour growth control. RESULTS: A dose-dependent bLAC-inducing apoptotic-like cell death was shown in UCC cells tested, including cells refractory to classic apoptosis-inducing agents, whereas bLA showed little cytotoxicity. bLAC selectively destroyed cancer cells in spheroids. Intravesical bLAC therapy demonstrated marked reduction in tumour growth/progression and significantly prolonged animal survival vs saline instillations (P = 0.004, log-rank test) and showed comparable efficacy with BCG (standard) therapy. CONCLUSION: The findings identify bLAC as a new candidate for UCC therapy and suggests that topical administration of bLAC alone or with BCG to prevent progression of bladder cancer warrants further exploration.

Effects of different fractions of whey protein on postprandial lipid and hormone responses in type 2 diabetes.

BACKGROUND/OBJECTIVES: Exacerbated postprandial lipid responses are associated with an increased cardiovascular risk. Dietary proteins influence postprandial lipemia differently, and whey protein has a preferential lipid-lowering effect. We compared the effects of different whey protein fractions on postprandial lipid and hormone responses added to a high-fat meal in type 2 diabetic subjects. SUBJECTS/METHODS: A total of 12 type 2 diabetic subjects ingested four isocaloric test meals in randomized order. The test meals contained 100 g of butter and 45 g of carbohydrate in combination with 45 g of whey isolate (iso-meal), whey hydrolysate (hydro-meal), α-lactalbumin enhanced whey (lac-meal) or caseinoglycomacropeptide enhanced whey (CGMP-meal). Plasma concentrations of triglyceride, retinyl palmitate, free fatty acid, insulin, glucose, glucagon, glucagon-like peptide 1 and glucose-dependent insulinotropic peptide were measured before and at regular intervals until 8-h postprandially. RESULTS: We found no statistical significant differences between meals on our primary variable triglyceride. The retinyl palmitate response was higher after the hydro-meal than after the iso- and lac-meal in the chylomicron-rich fraction (P=0.008) while no significant differences were found in the chylomicron-poor fraction. The hydro- and iso-meal produced a higher insulin response compared with the lac- and CGMP-meal (P<0.001). Otherwise no significant differences in the hormone responses were found in the incremental area under the curve over the 480-min period. CONCLUSIONS: A supplement of four different whey protein fractions to a fat-rich meal had similar effects on postprandial triglyceride responses in type 2 diabetic subjects. Whey isolate and whey hydrolysate caused a higher insulin response.

An α-lactalbumin-enriched and symbiotic-supplemented v. a standard infant formula: a multicentre, double-blind, randomised trial.

The aim of the present study was to evaluate the safety, tolerance and preventive effect on atopic dermatitis of an experimental α-lactalbumin-enriched and symbiotic-supplemented infant formula. A total of ninety-seven non-breastfed term neonates were enrolled into a double-blind, multicentre, randomised controlled trial in which they received experimental (n 48) or standard formula (n 49) for 6 months. The primary outcome was weight at 6 months of age. Secondary outcomes were gastrointestinal tolerance and manifestation of atopic dermatitis. Faecal secretory IgA (SIgA) concentration and microbiota composition of forty-three infants were analysed at 1 and 6 months. Growth was similar in both groups. At 1 month, compared to those in the control group, infants in the experimental group exhibited less crying or agitation, and more quiet behaviour (P=0·03). At 6 months, atopic dermatitis was less frequently observed in the experimental group (P<0·05). Decrease of faecal SIgA concentration between 1 and 6 months was mainly observed in the control group. This decrease was significantly associated with atopic dermatitis (P<0·014) and negatively correlated to the level of colonisation by bifidobacteria (P<0·005). In conclusion, compared to the control formula, the experimental formula guaranteed a similar growth, was better tolerated at 1 month and had a protective effect against the development of atopic dermatitis.

Preclinical activity profile of α-lactoalbumin, a whey protein rich in tryptophan, in rodent models of seizures and epilepsy.

PURPOSE: To evaluate the potential anticonvulsant activity of α-lactalbumin (ALAC), a whey protein rich in tryptophan (TRP) relative to other large neutral amino acids (LNAAs), in rodent models of seizures and epilepsy. METHODS: The effects of ALAC administered per os were evaluated by standard protocols against audiogenic seizures in Genetic Epilepsy Prone Rats (GEPR-9 rats), maximal electroshock (MES)-induced seizures in rats, pilocarpine-induced seizures in mice, spontaneous chronic seizures in mice exposed to pilocarpine-induced status epilepticus (SE), and absence seizures in WAG/Rij rats. In some models, carbamazepine (CBZ) was included as an active control. Plasma TRP/LNAAs ratios were measured by GC-MS. RESULTS: Single doses of ALAC up to 500 or 6000 mg/kg were devoid of anticonvulsant activity in all models tested. Conversely, 5- and 12-day treatment with ALAC (250-1000 mg/kg/day) in GEPR rats reduced dose-dependently seizure scores and prolonged latency to clonus onset, with full persistence of the effect for up to 12h. ALAC (125-500 mg/kg/day for 15 days) protected against seizures induced by 250 mg/kg pilocarpine, but was less effective against higher pilocarpine doses. Similarly to CBZ, ALAC (125-500 mg/kg/day for 15 days) was also effective against spontaneous seizures in the post-pilocarpine SE model. ALAC (up to 6000 mg/kg/day for 12 days) did not prevent MES-induced seizures, although it reduced the duration of tonic extension at doses between 250 and 1000 mg/kg/day. Absence seizures in WAG/Rij rats were not significantly affected by ALAC. Plasma TRP/LNAAS ratios increased 2- to 3-fold after dosing with ALAC (250 mg/kg/day) for 7 and 14 days, respectively. CONCLUSIONS: ALAC exerts significant protective activity against seizures in animal models, the effect being especially prominent against audiogenic seizures in GEPR-9 rats, seizures induced by low-dose pilocarpine in mice, and spontaneous seizures in mice exposed to pilocarpine-induced SE. This action is likely to be mediated by increased availability of TRP in the brain, with a consequent increase in 5-HT mediated transmission.

Naturally occurring, tumor-specific, therapeutic proteins.

The emerging approach to cancer treatment known as targeted therapies offers hope in improving the treatment of therapy-resistant cancers. Recent understanding of the molecular pathogenesis of cancer has led to the development of targeted novel drugs such as monoclonal antibodies, small molecule inhibitors, mimetics, antisense and small interference RNA-based strategies, among others. These compounds act on specific targets that are believed to contribute to the development and progression of cancers and resistance of tumors to conventional therapies. Delivered individually or combined with chemo- and/or radiotherapy, such novel drugs have produced significant responses in certain types of cancer. Among the most successful novel compounds are those which target tyrosine kinases (imatinib, trastuzumab, sinutinib, cetuximab). However, these compounds can cause severe side-effects as they inhibit pathways such as epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor, which are also important for normal functions in non-transformed cells. Recently, a number of proteins have been identified which show a remarkable tumor-specific cytotoxic activity. This toxicity is independent of tumor type or specific genetic changes such as p53, pRB or EGFR aberrations. These tumor-specific killer proteins are either derived from common human and animal viruses such as E1A, E4ORF4 and VP3 (apoptin) or of cellular origin, such as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and MDA-7 (melanoma differentiation associated-7). This review aims to present a current overview of a selection of these proteins with preferential toxicity among cancer cells and will provide an insight into the possible mechanism of action, tumor specificity and their potential as novel tumor-specific cancer therapeutics.

Modulation of mammary gland development in pre-pubertal mice as affected by soya and milk protein supplements.

The objective of the present study was to determine the effects of soya and whey milk protein, α-lactalbumin (α-LA), on mammary gland morphology and the structural support of the gland, in pre-pubertal mice after 7 d of treatment. In Expt 1, weaned (day 21) CD1 mice were given one of the four treatments, three included dietary supplements: (1) control diet, casein, (2) soya, (3) α-LA and (4) subcutaneous injection of 2·5 µg oestradiol benzoate in 20 µl maize oil and fed the control diet. All diets were isoenergetic with equal protein concentrations. All groups that were not treated with oestradiol received the vehicle. Whole-mount analyses were performed to determine longitudinal ductal growth and terminal end bud development. DNA was extracted from the gland and assessed by spectrophotometry (260/280 nm). Tissue extracts for extracellular matrix (ECM) proteins, matrix metalloproteinase-2 (MMP(2)), tissue inhibitor of MMP(2) (TIMP(2)), and serum oestradiol and mammary tissue epidermal growth factors (EGF) were measured by immunoassays. Expt 2 utilised the Her2/neu transgenic strain, with the same protocols. Statistical significance was determined by one-way ANOVA. From Expt 1 and 2, soya and α-LA significantly increased ductal elongation when compared with the oestrogen and control groups. These results were corroborated by data on total DNA and the ratio of MMP(2):TIMP(2). The ratio of MMP(2):TIMP(2) was affected by α-LA. Serum oestradiol was decreased only in the oestradiol-treated groups in both experiments. Soya is known to be oestrogenic and can act on epithelia directly. The mechanism by which α-LA affects glandular development is by modulating the ECM or by promoting the synthesis/activity of EGF.

Structure and function of human α-lactalbumin made lethal to tumor cells (HAMLET)-type complexes.

Human α-lactalbumin made lethal to tumor cells (HAMLET) and equine lysozyme with oleic acid (ELOA) are complexes consisting of protein and fatty acid that exhibit cytotoxic activities, drastically differing from the activity of their respective proteinaceous compounds. Since the discovery of HAMLET in the 1990s, a wealth of information has been accumulated, illuminating the structural, functional and therapeutic properties of protein complexes with oleic acid, which is summarized in this review. In vitro, both HAMLET and ELOA are produced by using ion-exchange columns preconditioned with oleic acid. However, the complex of human α-lactalbumin with oleic acid with the antitumor activity of HAMLET was found to be naturally present in the acidic fraction of human milk, where it was discovered by serendipity. Structural studies have shown that α-lactalbumin in HAMLET and lysozyme in ELOA are partially unfolded, 'molten-globule'-like, thereby rendering the complexes dynamic and in conformational exchange. HAMLET exists in the monomeric form, whereas ELOA mostly exists as oligomers and the fatty acid stoichiometry varies, with HAMLET holding an average of approximately five oleic acid molecules, whereas ELOA contains a considerably larger number (11- 48). Potent tumoricidal activity is found in both HAMLET and ELOA, and HAMLET has also shown strong potential as an antitumor drug in different in vivo animal models and clinical studies. The gain of new, beneficial function upon partial protein unfolding and fatty acid binding is a remarkable phenomenon, and may reflect a significant generic route of functional diversification of proteins via varying their conformational states and associated ligands.